O2-arylated or O2-glycosylated 1-substituted diazen-1-ium-1,2-diolates and O2-substituted 1-[(2-carboxylato) pyrrolidin-1-yl]diazen-1-ium-1,2-diolates

ABSTRACT

Diazeniumdiolates, wherein the N 1  position is substituted by an inorganic or organic moiety and the O 2 -oxygen is bound to a substituted or unsubstituted aromatic group, are provided. Also provided are O 2 -glycosylated 1-substituted diazen-1-ium-1,2-diolates (O 2 -glycosylated diazeniumdiolates) and O 2 -substituted 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates (1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates). The O 2 -aryl diazeniumdiolates are stable with respect to the hydrolytic generation of nitric oxide in neutral to acidic solutions and generate nitric oxide in basic or nucleophilic environments or microenvironments. Also provided are compositions, including pharmaceutical compositions, comprising such compounds and methods of using such compounds.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to O²-aryl 1-substituteddiazen-1-ium-1,2-diolates (O²-aryl diazeniumdiolates) O²-glycosylated1-substituted diazeniumdiolates, and O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates, compositionscomprising such diazeniumdiolates, methods of using suchdiazeniumdiolates, and methods of preparing O²-aryl diazeniumdiolates.

BACKGROUND OF THE INVENTION

[0002] Nitric oxide (NO) has been implicated in a wide variety ofbioregulatory processes, and compounds, which contain nitric oxide orare capable of releasing nitric oxide, have been identified as useful inregulating these processes. Many classes of nitric oxide-containingand/or -releasing adducts are known in the art, such as glyceryltrinitrate and nitroprusside (reviewed in U.S. Pat. No. 5,405,919(Keefer et al.), including limitations of their use in biologicalapplications). The limited utility of such compounds has, in part, givenrise to the development of another class of nitric oxide-generatingcompounds, diazeniumdiolates, which are especially useful biologically.

[0003] Diazeniumdiolates include compounds containing an N₂O₂ ⁻functional group and are structurally and functionally distinct fromnitrosamines (see, e.g., Reilly, U.S. Pat. No. 3,153,094). The knowndiazeniumdiolates are disclosed in recently issued patents. U.S. Pat.Nos. 5,039,705 (Keefer et al.) and 5,208,233 (Keefer et al.) disclosesecondary amine-nitric oxide adducts and salts thereof. U.S. Pat. Nos.5,155,137 (Keefer et al.) and 5,250,550 (Keefer et al.) disclosecomplexes of nitric oxide and polyamines. U.S. Pat. No. 5,389,675(Christodoulou et al.) discloses mixed ligand metal complexes of nitricoxide-nucleophile adducts and U.S. Pat. Nos. 5,525,357 (Keefer et al.)and 5,405,919 (Keefer et al.) disclose polymer-bound nitricoxide/nucleophile adduct compositions. U.S. Pat. Nos. 4,954,526 (Keeferet al.; the '526 patent) and 5,212,204 (Keefer et al.) disclose the useof ionic diazeniumdiolates as cardiovascular agents. In addition, the'526 patent discloses O²-substituted and metal-bound diazeniumdiolates.Keefer et al., U.S. Pat. No. 5,366,997 ('997), disclosesdiazeniumdiolates having the formula:

[0004] in which the O²-oxygen of the N₂O₂ ⁻ group is bonded to thefunctional group R³. When the R³ group is cleaved from the O²-oxygen, NOcan be released spontaneously.

[0005] Although Keefer et al. ('997) discloses that (i) R¹ and R²,together with the nitrogen atom to which they are bonded, can form apyrrolidinyl, piperazino or other heterocyclic group, (ii) R³ is a C₁₋₁₂straight-chain or C₃₋₁₂ branched-chain alkyl, optionally olefinic and/orsubstituted with hydroxy, halo, acyloxy or alkoxy, a C₁₋₁₂unsubstituted/substituted acyl, sulfonyl, carboxamido, sulfinyl,sulfenyl, a carbonate derivative or a carbamate derivative, and (iii)the pyrrolidinyl group can have the structure:

[0006] wherein w=4, and R⁴=hydrogen, a C₁₋₈ straight or branched chainalkyl, a C₃₋₈ cycloalkyl, or a substituted or an unsubstituted aryl,Keefer et al. ('997) does not disclose that R³ is an aryl or asubstituted aryl or that the pyrrolidino group can be substituted with asubstituted or unsubstituted carboxyl group (see, also, Example 1 ofU.S. Pat. No. 5,632,981) at position 2. Similarly, Keefer et al. ('997)does not disclose O²-glycosylation of diazeniumdiolates.

[0007] Heretofore it was not known that O²-aryl substitutions of thediazeniumdiolates was possible. Further, chemical studies of previouslydisclosed diazeniumdiolates led to the conclusion that they aregenerally at least as stable at high pH as they are at low pH, and that,unlike certain other classes of “nitrovasodilator” drugs, their rates ofNO release are not affected by the presence of nucleophilic thiols.

[0008] Thus, there remains a need for such classes of diazeniumdiolates,which offer advantages over other currently available diazeniumdiolates.In this regard, the O²-aryl substituted diazeniumdiolates areadvantageous in that they can release NO spontaneously under alkalineconditions or after nucleophilic attack. O²-Aryl substituteddiazeniumdiolates also can release NO spontaneously after a combinationof oxidative or electophilic activation and nucleophilic attack.

[0009] It is, therefore, a principal object of the present invention toprovide a nitric oxide/nucleophile adduct in which the O²-oxygen of theN₂O₂ ⁻ group is derivatized with an aryl or substituted aryl group toprotect the diazeniumdiolate against the spontaneous release of NO. Itis another object of the invention to provide a novel class ofdiazeniumdiolates, which resists releasing nitric oxide in neutral oracidic solutions, but releases NO on nucleophilic attack or onincreasing the pH. It is still another object of the present inventionto provide O²-glycosylated 1-substituted diazen-1-ium-1,2-diolates andO²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates. It is afurther object of the present invention to provide compositionscomprising such compounds, including compositions comprising a nitricoxide/nucleophile adduct comprising a novel targeting moiety. It is arelated object to provide O²-aryl substituted diazeniumdiolates, whichare amenable to biological tissue-targeting strategies, which offergreater flexibility and specificity for targeting NO release. It is astill further object of the present invention to provide methods ofusing such compounds. These and other objects of the present invention,as well as additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides an O²-aryl substituteddiazeniumdiolate (i.e., O²-aryl diazeniumdiolate) illustrated by theformula:

[0011] wherein X is an inorganic or organic moiety and Q is an arylmoiety. In this novel class of compounds an atom of the aryl ring moietyQ is bonded to the O²-oxygen of the N₂O₂ ⁻ functional group. Thediazeniumdiolates of Formula (I) are stable with respect to thehydrolytic generation of nitric oxide in neutral to acidic solutions.Surprisingly, these novel compounds, or the resultant product of thesecompounds after oxidative or electrophilic activation, have provencapable of generating nitric oxide in basic or nucleophilicenvironments, in which the aryl moiety is separated from the remainderof the diazeniumdiolate.

[0012] The present invention also provides O²-glycosylated 1-substituteddiazen-1-ium-1,2-diolates and O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl)diazen-1-ium-1,2-diolates, both ofwhich can be represented by the formula:

[0013] in which X and R are organic and/or inorganic moieties as definedherein, although for O²-glycosylated diazeniumdiolates, R must be asaccharide.

[0014] Further with respect to the O²-glycosylated 1-substituteddiazen-1-ium-1,2-diolates, the moiety X can be any organic or inorganicgroup. Preferably, X contains atoms other than carbon and hydrogen, andis linked to the nitrogen of the diazeniumdiolate through an atom otherthan carbon. Most preferably, X is an amino group, and is linked to thenitrogen of the diazeniumdiolate through a nitrogen atom.

[0015] With respect to the O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates, X ofFormula Ia can be

[0016] such that the [1-(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolatescan be structurally represented by the formula:

[0017] wherein R²² is hydrogen, hydroxyl, OM, wherein M is a cation,halo, or X¹R²³R²⁴, wherein X¹ is oxygen, nitrogen or sulfur and R²³ andR²⁴ are independently a substituted or unsubstituted C₁₋₂₄ alkyl, asubstituted or unsubstituted C₃₋₂₄ cycloalkyl, a substituted orunsubstituted C₂₋₂₄ olefinic, a substituted or unsubstituted aryl (suchas acridine, anthracene, benzene, benzofuran, benzothiophene,benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll,cinnoline, furan, imidazole, indole, isobenzofuran, isoindole,isoxazole, isothiazole, isoquinoline, naphthalene, oxazole,phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide,phthalazine, phthalocyanine, porphin, pteridine, purine, pyrazine,pyrazole, pyridazine, pyridine, pyrimidine, pyrrocoline, pyrroleiquinolizinium ion, quinoline, quinoxaline, quinazoline, sydnone,tetrazole, thiazole, thiophene, thyroxine, triazine, and triazole), or aheterocyclic group, such as glycosyl, and the like, and when X¹ is O orS, there is no R²⁴ group. Alternatively, when X¹ is nitrogen, R²³ andR²⁴, together with X¹, form a heterocyclic ring, such as a heterocyclicring selected from the group consisting of:

[0018] in which A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸,R⁹, R²⁵, and R²⁶ are hydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈branched chain alkyl, a C₃₋₈ cycloalkyl, or an aryl. The aforementionedR²³ and R²⁴ groups can be unsubstituted or substituted as appropriate.For example, the R²³ and R²⁴ groups can be substituted as appropriatewith acyloxy, acylthio, hydroxyl, amino, carboxyl, mercapto, halo,amido, sulfonyl, sulfoxy, sulfenyl, phosphono, phosphate, and the like.

[0019] Further with respect to the O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates, the moietyR of Formula Ia can be any organic or inorganic moiety, which iscovalently bound to the terminal oxygen of the diazeniumdiolate as shownbut which is other than hydrogen and is a substituted or unsubstitutedC₁₋₁₂ straight chain or C₃₋₁₂ branched chain alkyl, a substituted orunsubstituted C₂₋₁₂ straight chain or C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted C₁₋₁₂ acyl, sulfonyl, carboxamido, aglycosyl group, an aryl group, or a group of the formula—(CH₂)_(n)—ON═N(O)NR²⁸R²⁹, wherein n is an integer of 2-8, and R²⁸ andR²⁹ are independently a C₁₋₁₂ straight chain alkyl, a C₃₋₁₂ branchedchain alkyl, a C₂₋₁₂ straight chain or C₃₋₁₂ branched chain olefinic, orR²⁸ and R²⁹, together with the nitrogen atom to which they are bonded,form a heterocyclic group, preferably a pyrrolidino, piperidino,piperazino or morpholino group. The aforementioned R groups can beunsubstituted or substituted. Preferred substitutions include those madewith hydroxy, halo, acyloxy, alkoxy, acylthio, or benzyl.

[0020] In another aspect, the present invention comprises a composition,including a pharmaceutical composition, comprising a present inventivediazeniumdiolate. The pharmaceutical composition preferably additionallycomprises a pharmaceutically acceptable carrier.

[0021] In yet another aspect, the present invention provides methods ofusing a compound in accordance with the present invention.

[0022] In still another aspect, the present invention provides a methodof making O²-aryl diazeniumdiolates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a graph of Trp37 fluorescence (RFU) versus time (min),which depicts zinc ejection from HIV-1 nucleocapsid p7 protein byO²-aryl diazeniumdiolates. In the graph, ◯ represents the negativecontrol, i.e., no drug, □ represents the positive control, i.e., 624151(See Rice et al., Antimicrob. Agents Chemother. 41: 419426 (1997)), ▪represents the compound of Example 1 (LK1), ♦ represents the compound ofExample 8 (LK2), ▴ represents the compound of Example 5 (LK3), •represents the compound of Example 10 (LK4), and X represents thecompound of Example 11 (LK5).

[0024]FIG. 2 is a graph of relative NO release rate versus time (min),which depicts the catalysis of NO release from DNP-PYRRO/NO byglutathione S-transferase (GST).

DETAILED DESCRIPTION OF THE INVENTION O²-arylated diazeniumdiolates

[0025] The present invention provides an O²-aryl 1-substituteddiazeniumdiolate (i.e., O²-aryl 1-substituted diazen-1-ium-1,2-diolate)having the formula:

[0026] wherein X is an organic or inorganic moiety and Q is an arylgroup.

[0027] In accordance with the invention, the O²-oxygen of the N₂O₂ ⁻group is bonded directly to an atom of the ring of the aryl group.Stated another way, there are no spacer atoms (e.g., methylene) thatseparate the O²-oxygen from the aryl ring. If the aryl group comprises abicyclic or polycyclic moiety and all rings of the aryl group are notaromatic, then the linkage between the O²-oxygen and the aryl group isthrough an atom that is part of an aromatic ring. Further, the O²-oxygencan be linked to any aromatic ring atom of the aryl group that iscapable of bonding to the O²-oxygen of the N₂O₂ ⁻ group. Atoms of thearomatic ring that are capable of bonding with the O²-oxygen of the N₂O₂⁻ group are typically carbon and nitrogen, although there can be otherlinkages as well.

[0028] While not wishing to be bound to any particular theory, it ispresently believed that the bonding of the O²-oxygen with the atom ofthe aryl ring is accomplished by bonding to an activated atom of thering. Activation can be accomplished through any suitable mechanism. Inthis regard, a preferred mechanism of activating an aryl ring is byreacting the diazeniumdiolate through an atom of the aryl ringpossessing a partial positive charge or, more specifically, bydisplacing an amino substituent of the ring structure.

[0029] In the first preferred reaction mechanism, the aryl ring issubstituted by a suitable electron-withdrawing group(s), which can bepart of the ring, as in Example 12, and a “leaving group” prior toreaction with the diazeniumdiolate. It will be appreciated by thoseskilled in the art that the electron-withdrawing group and the leavinggroup can, in some instances, be the same moiety. The leaving group isdisplaced by the diazeniumdiolate to form the O²-aryl diazeniumdiolateof the present invention. Suitable leaving groups include, but are notlimited to, F, Cl, Br, I, NO₂, OSO₂R, and OSO₃R, wherein R is an organicmoiety, a metal center, or the like, the composition of which is wellunderstood by those skilled in the art. By way of illustration and notin limitation, suitable R groups include H, alkyl, alkenyl, or aryl.This reaction mechanism is based on the well known S_(N)Ar mechanism;for example, see Nucleophilic Aromatic Displacement: The Influence ofthe Nitro Group, Francois Terrier, VCH Publishing, Inc., New York, N.Y.,pages 1-11 (1991). Preferably, these S_(N)Ar reactions are carried outin electron-deficient aromatic rings comprising at least oneelectron-withdrawing group.

[0030] In the second preferred reaction mechanism, an aryl reactant issubstituted by a suitable amino group, which allows directderivatization (e.g., after diazotization of the amino group) of thering atom of the aryl group that is bound to the displaced amino group.There is no requirement for the atom of the aryl ring linked to theO²-oxygen to be activated after it has been incorporated into thepresent inventive compound. However, if this atom is activated afterbeing incorporated into the present inventive compound, then thediazeniumdiolate moiety to which it is bound may be displaced throughfurther nucleophilic displacement (e.g., in a suitably strong base).Alternatively, an oxidative or electrophilic activation event can alterthe present inventive compound so that the aryl ring atom linked to theO²-oxygen becomes activated, thereby rendering the compound subject tofurther nucleophilic displacement, as observed above.

[0031] Advantageously, the compounds of the present invention have newand useful properties, which are not possessed by other nitricoxide/nucleophile adducts previously known in the art. In general, thecompounds of the present invention are stable at neutral or acidic pH(i.e., at neutral or acidic pH, the compounds indicated by Formula I donot generate NO). Another advantageous property of the compounds of thepresent invention is that the O²-aryl linkage is often susceptible tocleavage by nucleophiles, including hydroxide ions. When the typicalO²-aryl diazeniumdiolate or the oxidatively or electrophilicallyactivated O²-aryl diazeniumdiolate of the present invention is placedinto a basic or nucleophilic environment, the aryl linkage to theO²-oxygen can be broken. The resulting diazeniumdiolate ionspontaneously degrades via a predictable, first order mechanism, givingrise to NO. The resulting aryl group is substituted with a nucleophileprovided by the environment. If the nucleophile provided by theenvironment is part of an enzyme, that enzyme can be inactivated. Thesusceptibility to nucleophilic attack of the O²-aryl diazeniumdiolatesalso makes them particularly amenable to designing prodrugs fortargeting nitric oxide to nucleophilic tissue components, body sites andmicroenvironments in the body.

[0032] The compounds of the present invention are also useful toidentify and quantify individual thiols (organic —SH containingcompounds) when the thiols are present in mixtures. For example, asample suspected to consist of C₄-C₈ straight-chain thiols can beanalyzed by dissolving the product of Example 1 in tetrahydrofuran oranother inert solvent, then mixing a molar excess of the resultingsolution with the sample to be assayed. After the ensuing reaction iscomplete, an aliquot is subjected to HPLC analysis using an ultravioletdetection system. Peaks found in the resulting chromatogram can beidentified by comparing their retention times to those of independentlyderivatized authentic standards of the individual C₄-C₈ straight-chainthiols, and quantified by transforming peak areas to concentrations viathe individual standard curves.

[0033] With respect to the O²-aryl diazeniumdiolates, “aryl group” asused herein refers to any aromatic group, regardless of whether it ispart of a (homo)cyclic, heterocylic, or polycyclic structure. Thestandard understanding of “aromatic” is used herein (See, e.g., L. G.Wade, Jr., Organic Chemistry, 2d Edition, Prentice Hall, EnglewoodCliffs, N.J., 682-683 (1991)). The aryl group, as used herein, can alsohave a wide variety of substituent groups. Any suitable aryl substituentcan be used providing that the substituent does not destroy thearomaticity of the aryl ring.

[0034] Turning to the aryl group Q of Formula I, Q is intended toinclude all aryl groups that are (or can be made) amenable to reactionwith the O²-oxygen atom of a diazeniumdiolate. The moiety Q thusincludes homocyclic, heterocyclic, and polycyclic aromatic structures aswell as derivatives thereof. Illustrative of the aryl groups Q areacridine, anthracene, benzene, benzofuran, benzothiophene, benzoxazole,benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan,imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole,isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine,phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine,porphin, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrocoline, pyrrole, quinolizinium ion, quinoline,quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene,thyroxine, triazine, and triazole.

[0035] In keeping with the invention, each of these aromatic compounds Qcan be variably derivatized with the numerous substituents well known inthe art that are capable of being substituted into an aromatic ring solong as the aromaticity of the ring is maintained. For example, thesubstituents of the aryl moiety, Q, can include X[N(O)NO]⁻, wherein X isas defined hereinafter and is the same as X of Formula I, halo, hydroxy,alkylthio, arylthio, alkoxy, aryloxy, amino, mono- or di-substitutedamino, ammonio or substituted ammonio, nitroso, cyano, sulfonato,mercapto, nitro, oxo, C₁-C₂₄ aliphatic, C₃-C₁₂ olefinic, C₃-C₂₄cycloalkyl, C₃-C₂₄ heterocycloalkyl, benzyl, phenyl, substituted benzyl,substituted phenyl, benzylcarbonyl, phenylcarbonyl, saccharides,substituted benzylcarbonyl, substituted phenylcarbonyl and phosphorusderivatives. Illustrative phosphorus derivatives include phosphate andphosphono moieties. Illustrative phosphate moieties include (OH)₂P(O)O—and substituted (OH)₂P(O)O-moieties, wherein one or more oxygen atomscan be independently replaced by S or NR′, wherein R′ is understood tobe a C₁-C₁₀ containing aliphatic, cycloalkyl, or aryl group.Illustrative C₁-C₂₄ aliphatic substituents comprise C₁-C₂₄ acyl, and

[0036] wherein R is hydrogen, substituted or unsubstituted C₁-C₂₃aliphatic, substituted or unsubstituted C₃-C₂₃ cycloalkyl, substitutedor unsubstituted C₃-C₁₂ olefinic, benzyl, phenyl, substituted benzyl orsubstituted phenyl, and said substituted benzyl or substituted phenyl issubstituted with one to five substituents selected from the groupconsisting of nitro, halo, hydroxy, C₁-C₂₄ alkyl, C₁-C₂₄ alkoxy, amino,mono-C₁-C₂₄ alkylamino, di-C₁-C₂₄ alkylamino, cyano, phenyl and phenoxy.Preferred saccharides include ribose, glucose, deoxyribose, dextran,starch, glycogen, lactose, fucose, galactose, fructose, glucosamine,galactosamine, heparin, mannose, maltose, sucrose, sialic acid, andcellulose. Other preferred saccharides are phosphorylated,3,5-cyclophosphorylated, and polyphosphorylated hexoses and pentoses.

[0037] Examples of substituted aryl compounds of the present inventionthat can be linked to the diazeniumdiolate group comprise dinitrophenol(a benzene), hypoxanthine (a purine), uridine (a pyrimidine), vitaminK₅(a naphthalene) and ribosyl uridine (a nucleoside).

[0038] In another particular embodiment of the present invention, thearyl moiety is identical to or structurally analogous to molecules, orsubstituents thereof, normally found in living organisms. Thesebiologically relevant groups can be selected from nucleotides,nucleosides, and nucleic acids, peptides, including peptide hormones,non-peptide hormones, vitamins and other enzyme cofactors such asporphyrins, and others. Examples of biologically relevant aryl groupsare thyroxine, NAD (or NADH), chlorophyll, hypoxanthine, uridine, andvitamin K₅.

[0039] The following reaction schematics illustrate methods of preparingthe O²-aryl diazeniumdiolates of the present invention. In theseillustrative reactions, in general, a solution of a diazeniumdiolate(X—[N₂O₂ ⁻]) in 5% aqueous sodium bicarbonate (which is weakly basic) iscooled to 0° C., preferably under a blanket of inert gas such asnitrogen. A solution containing one equivalent of the activated aromaticreagent in a solvent, such as t-butyl alcohol, dimethyl sulfoxide, orN,N-dimethylformamide, is then added slowly. While not being bound toany particular theory, it is believed that polar non-protic solvents arepreferred. The reaction temperature is raised slightly for less reactivearyl moieties, for example, to ambient temperatures or higher.Generally, a precipitate forms upon addition. The mixture is thenallowed to warm to room temperature gradually and stirred overnight. Theproduct may be extracted with a suitable extraction agent, such asdichloromethane, and washed subsequently with cold dilute hydrochloricacid and then with sodium bicarbonate solution. The organic layer isdried over a suitable drying agent, such as sodium sulfate, filtered,preferably through a layer of anhydrous magnesium sulfate, andevaporated under vacuum to give the crude product. Usually, the productis solid. Recrystallization from ethanol or other suitable solvents is apreferred method of purifying the product. It will be appreciated by oneskilled in the art that these conditions can be modified to suit theparticular application of the artisan. Accordingly, alternative methodsof preparation are also embraced.

[0040] Chlorinated quinoline and isoquinoline can be reacted with adiazeniumdiolate such that the Cl substituent is replaced by theO²-oxygen of a diazeniumdiolate, as shown below:

[0041] Additionally, quinazoline can be incorporated as shown:

[0042] The phthalazines also are incorporated in accordance with thepresent invention, as indicated:

[0043] Acridine can be incorporated as indicated:

[0044] Cinnoline can also be incorporated as indicated:

[0045] Quinoxaline can also be incorporated as indicated:

[0046] Oxygen- and sulfur-containing heteroaromatics can also be used asthe aromatic reagent for O²-oxygen substitution of the diazeniumdiolatein accordance with the present invention. For example, oxazole andbenzoxazole can be derivatized at the 2-position as indicated:

[0047] Similarly, thiazole and benzothiazole can also be derivatized atthe 2-position.

[0048] A derivatized Vitamin K₇ can also be prepared, as indicated:

[0049] The O²-diazeniumdiolated atom of the aryl ring in the right-most(directly above) structure is not activated. Therefore, the right-moststructure is resistant to nucleophilic attack, which would re-generateX—N₂O₂ ⁻, which, in turn, would spontaneously degrade to produce NO.Therefore, the right-most structure must undergo oxidative preactivationprior to nucleophilic attack in order to generate NO. This oxidativepreactivation requirement would also be of advantage in targeting a cellor organ type that is uniquely able to perform the required oxidation,thereby limiting NO exposure to the desired tissue while avoidingexposure at other NO— sensitive portions of the anatomy.

[0050] Illustrative of the class of compounds requiring electrophilicpreactivation is the compound indicated below:

[0051] Triazines can likewise be the aromatic reagent that forms theO²-aryl substituted diazeniumdiolates of the present invention as shownbelow. The synthesis of such compounds should enhance the potency ofexisting triazine-derived herbicides.

[0052] Nucleic acids and the nitrogenous bases they comprise (includingribosylated bases) can also be used as the aromatic reagent to form theO²-aryl substituted diazeniumdiolates of the present invention. This isillustrated in Example 13.

[0053] Another interesting O²-arylated diazeniumdiolate is the one shownas the product in the reaction below; it can co-generate NO andallopurinol on hydrolysis.

[0054] Advantageously, allopurinol is already known to bepharmaceutically useful. Thus, by converting known pharmaceuticallyuseful compounds containing a suitable aryl group to the O²-aryldiazeniumdiolates of the present invention, the present invention allowsexisting drugs to be enhanced by the release of NO.

[0055] Similarly, a derivative of a biopterin diazeniumdiolate can beprepared from a substituted pteridine, as indicated below.

[0056] An example of a suitable aryl substitution that utilizes linkagethrough a heteroatom is shown in the following scheme which can beeffected by reaction with BuONO or other suitable nitrosating agents.

[0057] A structural analog of Bendazac, a well-known anti-inflammatoryagent, can be prepared as indicated:

[0058] In accordance with the invention, any of the compounds in theclass of compounds defined as diazeniumdiolates can be subjected toO²-aryl substitution. Thus, for the compounds having Formula I, X can beany organic or inorganic moiety. Preferably, X contains atoms other thancarbon and hydrogen, and is linked to the nitrogen of the N₂O₂ ⁻ groupthrough an atom other than carbon. Most preferably, X is an amine, andis linked to the nitrogen of the N₂O₂ ⁻ group through a nitrogen atom.Suitable moieties of X also include, but are not limited to, C₁-C₂₄aliphatic, aryl, and nonaromatic cyclic. By “aliphatic” is meant acyclicmoieties containing carbon and hydrogen and optionally containingnitrogen, oxygen, sulfur, phosphorus, and halogens. By “aryl” is meant,as hereinabove, a moiety containing at least one aromatic ring.Preferably, the aryl moiety is a C₃-C₃₀-containing moiety. Bynonaromatic cyclic is meant a moiety containing at least one ringstructure and no aromatic rings. Preferably, the non-aromatic cyclicmoiety is a C₃-C₃₀-containing moiety.

[0059] The moiety X of Formula I can be unsubstituted or substitutedwith suitable additional moieties, such as, for example, —[N(NO)O⁻],halo, hydroxy, alkylthio, alkoxy, aryloxy, amino, mono- ordi-substituted amino, cyano, sulfonato, mercapto, nitro, substituted orunsubstituted C₁-C₁₂ aliphatic, substituted or unsubstituted C₃-C₈cycloalkyl, substituted or unsubstituted C₃-C₈ heterocycloalkyl,substituted or unsubstituted C₃-C₁₂ olefinic, benzyl, phenyl,substituted benzyl, substituted phenyl, benzylcarbonyl, phenylcarbonyl,saccharides, substituted benzylcarbonyl, substituted phenylcarbonyl andphosphorus derivatives. Illustrative phosphorus derivatives includephosphato and phosphono moieties. Illustrative phosphato moietiesinclude (OH)₂P(O)O— and substituted (OH)₂P(O)O— moieties, wherein one ormore oxygen atoms can be independently replaced by S or NR′, wherein R′is understood to be a C₁-C₈-containing aliphatic, cycloalkyl, or arylgroup. Preferred C₁-C₁₂ aliphatic substituents comprise C₁-C₁₂ acyl, and

[0060] wherein R is C₁-C₁₀ substituted or unsubstituted aliphatic,C₃-C₁₁ olefinic, C₃-C₈ substituted or unsubstituted cycloalkyl, benzyl,phenyl, substituted benzyl or substituted phenyl, and said substitutedbenzyl or substituted phenyl is substituted with one or two substituentsselected from the group consisting of halogen, hydroxy, C₁-C₄ alkyl,C₁-C₄ alkoxy, amino, mono-C₁-C₄ alkylamino, di-C₁-C₄ alkylamino, phenyland phenoxy. Preferred saccharides and polysaccharides include ribose,glucose, deoxyribose, dextran, starch, glycogen, lactose, galactose,fructose, glucosamine, galactosamine, heparin, mannose, maltose,sucrose, sialic acid, and cellulose. Other preferred saccharides arephosphorylated, 3,5-cyclophosphorylated, and polyphosphorylated pentosesand hexoses.

[0061] In one embodiment of the invention, X is an inorganic moiety asdescribed in U.S. Pat. No. 5,212,204. Preferred embodiments of FormulaI, in which X is inorganic, are ³¹ O₃S— (sulfite) and —O⁻ (oxide).

[0062] In another embodiment of the present invention, X is a polyamineas defined in U.S. Pat. No. 5,155,137. Thus, the polyamine substitutedO²-aryl diazeniumdiolates have the formula

[0063] wherein Q is the same as the Q in Formula I and is defined asabove, b and d can be the same or different and are zero or one, R¹, R²,R³, R⁴, and R⁵ are the same or different and comprise hydrogen,substituted or unsubstituted C₃-C₈ cycloalkyl, substituted orunsubstituted C₁-C₁₂ straight or branched chain alkyl, substituted orunsubstituted benzyl, substituted or unsubstituted benzoyl, substitutedor unsubstituted C₃-C₁₂ olefinic, phthaloyl, acetyl, trifluoroacetyl,p-toluyl, t-butoxycarbonyl, or 2,2,2-tri-halo-t-butoxycarbonyl. Thevalues of i, j, and k in Formula II can be the same or different and areintegers from 2 to 12.

[0064] In a preferred embodiment of the present invention the O²-aryldiazeniumdiolates are derived from the compounds disclosed in U.S. Pat.Nos. 5,039,705 (Keefer et al.) and 4,954,526 (Keefer et al.) and, thus,have the formula

[0065] wherein R⁶ and R⁷ can be the same or different and are chosenfrom H, C₁-C₁₂ straight chain alkyl, C₁-C₁₂ alkoxy or acyloxysubstituted straight chain alkyl, C₂-C₁₂ hydroxy or halo substitutedstraight chain alkyl, C₃-C₁₂ branched chain alkyl, C₃-C₁₂ hydroxy, halo,alkoxy, or acyloxy substituted branched chain alkyl, C₂-C₁₂ straightchain olefinic and C₃-C₁₂ branched chain olefinic, which areunsubstituted or which are substituted with hydroxy, alkoxy, acyloxy,halo or benzyl, provided that both R⁶ and R⁷ are not H; or R⁶ and R⁷,together with the nitrogen atom to which they are bonded, form aheterocyclic ring selected from the group consisting of:

[0066] wherein A is N, O, or S, w is 1 to 12, y is 1 or 2, z is 1 to 5,R⁸ is hydrogen, C₁-C₈ straight chain alkyl, C₃-C₈ branched chain alkyl,C₃-C₈ cycloalkyl, unsubstituted or substituted aryl, such as phenyl,tolyl or the like, and R⁹is hydrogen, C₁-C₆ straight chain alkyl orC₃-C₆ branched chain alkyl. Exemplary aza crown groups are1-aza-12-crown-4, 1-aza-15-crown-5, and 1-aza-18-crown-6. Where A isnitrogen, the nitrogen atom, itself, can be substituted, as described,for example, in U.S. application Ser. No. 08/475,732, which isincorporated by reference herein.

[0067] Further examples include the O²-aryl substituteddiazeniumdiolates derived from the compounds disclosed in U.S. Pat. No.5,250,550, previously incorporated in its entirety by reference, and,thus, have the formula wherein D is

[0068] and wherein R¹⁰ and R¹¹ are the same or different. Thesubstituents R¹⁰ and R¹¹ can be any suitable group, examples of whichinclude hydrogen, C₃-C₈ cycloalkyl, C₁-C₁₂ straight or branched chainalkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,t-butoxycarbonyl, and 2,2,2-trihalo-t-butoxycarbonyl. In Formula IV, fis an integer from 0 to 12.

[0069] Preferred O²-aryl substituted diazeniumdiolates also includethose of Example 14.

[0070] An alternative method of preparing O²-arylated diazeniumdiolatesis possible through adaptation of the following literature reaction(Stevens, J.Org. Chem. 29: 311-315 (1964)).

[0071] By substituting aryloxy anion ArO⁻ for the methoxide of Stevens'sreaction, it is possible to obtain O²-aryl diazeniumdiolates of variedstructure. Similarly, it is possible to obtain derivatives correspondingto ArS⁻ species.

O²-glycosylated diazeniumdiolates and1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates

[0072] The present invention also provides two other new classes ofdiazeniumdiolates, one class of which contains a hydrolytically labilegroup (R), which, upon cleavage to the free diazeniumdiolate (NO donor)X—NO═NO⁻, releases an innocuous and possibly beneficial saccharide andallows advantage to be taken of saccharide-based receptor-mediatedphenomena. The other class of diazeniumdiolates provides, among others,prodrugs of the salt disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO),which is an ultrafast NO donor of proven effectiveness as anantithrombotic agent and a vasodilator but is inherently extremelydifficult to derivatize, due to its instability (Saavedra et al., J.Med. Chem. 31:4361-4365 (1996); and U.S. Pat. No. 5,632,981 (Saavedra etal.)). The newly discovered ability to generate prodrugs of theultrafast NO donor PROLI/NO allows the PROLI/NO prodrugs to move freelythrough the circulatory system until they reach the desired organ orcell type for metabolic removal of the stabilizing O²-protecting group,thereby providing a rapid release of NO at the specific or preferredsite and obviating the need for administration by infusion at acontrolled rate in the vicinity of the target tissue. Additionally, thecorresponding nitrosamine, N-nitrosoproline, if formed in the biologicalmedium, does not pose a carcinogenic threat, unlike other nitrosamines.

[0073] Accordingly, the present invention provides O²-glycosylated1-substituted diazen-1-ium-1,2-diolates (O²-glycosylateddiazeniumdiolates) and O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates(1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates), both of which canbe represented by the formula:

[0074] In Formula Ia, X and R are organic and/or inorganic moieties asdefined herein.

O²-GLYCOSYLATED DIAZENIUMDIOLATES

[0075] With respect to the O²-glycosylated diazeniumdiolates, any of thecompounds in the class of compounds defined as diazeniumdiolates (seee.g., U.S. Pat. Nos. 5,039,705, 5,208,233, 5,155,137, 5,250,550,5,389,675, 5,525,357, 5,405,919 and related patents and patentapplications) can be subjected to O²-glycosylation, provided that the O²of the diazeniumdiolate is available for glycosylation. The moiety R ofFormula Ia can be any saccharide, which is attached to the O² of thediazeniumdiolate by the 2 position of a pyranose or furanose ring. Thesaccharide can be functionalized. Desirably, the saccharide and itsderivatives are hydrolyzable at physiological pH. The saccharide can bea monosaccharide, disaccharide, such as sucrose or maltose, anoligosaccharide or a polysaccharide. Preferred saccharides andpolysaccharides include, among others, ribose, glucose, deoxyribose,fucose, lactose, galactose, fructose, glucosamine, galactosamine,mannose, maltose, sucrose, and the many saccharide and oligosaccharideunits that serve as recognition sequences in receptor-mediated cellularinteractions. Other preferred saccharides include those that arephosphorylated, 3,5-cyclophosphorylated, and polyphosphorylated pentosesand hexoses.

[0076] By way of illustration, the saccharide residue (shown attached tothe diazeniumdiolate for illustrative purposes) can be an amino sugar,such as a glucosamine or a substituted glucosamine having the structure:

[0077] wherein R¹² and R¹³ can be the same or different and are ahydrogen, a C₁₋₆ alkyl, an acyl, a-phosphate, a sulfate, a peptide or aprotein. The saccharide residue can be, for example, glucuronic acid ora derivative thereof:

[0078] wherein R¹⁴ is X¹R¹⁵R¹⁶, wherein X¹ is N, O or S and, when X¹ isN, R¹⁵ and R¹⁶ are independently a hydrogen or a substituted or anunsubstituted C₁₋₂₄ alkyl, C₃₋₂₄ cycloalkyl, C₂₋₂₄ olefinic, aryl (suchas acridine, anthracene, benzene, benzofuran, benzothiophene,benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll,cinnoline, furan, imidazole, indole, isobenzofuran, isoindole,isoxazole, isothiazole, isoquinoline, naphthalene, oxazole,phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide,phthalazine, phthalocyanine, porphin, pteridine, purine, pyrazine,pyrazole, pyridazine, pyridine, pyrimidine, pyrrocoline, pyrrole,quinolizinium ion, quinoline, quinoxaline, quinazoline, sydnone,tetrazole, thiazole, thiophene, thyroxine, triazine, and triazole), orheterocyclic group, such as glycosyl and the like, and when X¹ is O orS, there is no R¹⁶ group.

[0079] Alternatively, when X¹ is nitrogen, R¹⁵ and R¹⁶ form aheterocyclic ring selected from the group consisting of:

[0080] wherein A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸ ishydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chain alkyl, aC₃₋₈ cycloalkyl, an aryl (such as phenyl, tolyl or the like), orcarboxylato and derivatives thereof as further described herein, and R⁹is hydrogen, a C₁₋₆ straight chain alkyl or a C₃₋₆ branched chain alkyl.The aforementioned groups can be unsubstituted or substituted asappropriate.

[0081] Exemplary aza crown groups (i.e., where A is N) are1-aza-12-crown-4, 1-aza-15-crown-5, and 1-aza-18-crown-6. Where A isnitrogen, the nitrogen atom, itself, can be substituted, as described,for example, in U.S. patent application Ser. No. 08/475,732.

[0082] Further with respect to the O²-glycosylated diazeniumdiolates,the moiety attached to the carbonyl group through X¹ can be anythingthat does not interfere with the cleavage to the diazeniumdiolate anion.

[0083] Further with respect to the O²-glycosylated diazeniumdiolates,the moiety attached to the carbonyl group through X¹ can be anythingthat does not interfere with the cleavage to the diazeniumdiolate anion.

[0084] Preferably, the moiety X contains atoms other than carbon andhydrogen, and is linked to the nitrogen of the N₂O₂ ⁻ group through anatom other than carbon. Most preferably, X is an amino group, and islinked to the nitrogen of the N₂O₂ ⁻ group through a nitrogen atom.Suitable moieties of X include, but are not limited to, C₁₋₂₄ aliphatic,aryl and non-aromatic cyclic groups. By “aliphatic” is meant an acyclicmoiety containing carbon and hydrogen and optionally containingnitrogen, oxygen, sulfur, phosphorus or a halogen. By “aryl” is meant amoiety containing at least one aromatic ring. Preferably, the arylmoiety is a C₆₋₃₀ moiety. By “non-aromatic cyclic” is meant a moietycontaining at least one ring structure and no aromatic rings.Preferably, the non-aromatic cyclic moiety is a C₆₋₃₀ moiety. Further, Xcan be unsubstituted or substituted with suitable additional moieties,such as, for example, —[N(NO)O⁻], a halo, a hydroxy, an alkylthio, analkoxy, an aryloxy, an amino, a mono- or di-substituted amino, a cyano,a sulfonato, a mercapto, a nitro, a substituted or unsubstituted C₁₋₁₂aliphatic, a substituted or unsubstituted C₃₋₈ cycloalkyl, a substitutedor unsubstituted C₃-C₁₂ olefinic, a substituted or unsubstituted C₃₋₈heterocycloalkyl, a benzyl, a phenyl, a substituted benzyl, asubstituted phenyl, a benzylcarbonyl, a phenylcarbonyl, a saccharide, asubstituted benzylcarbonyl, a substituted phenylcarbonyl and aphosphorus derivative. Illustrative phosphorus derivatives includephosphato and phosphono moieties. Illustrative phosphato moietiesinclude (OH)₂P(O)O— and substituted (OH)₂P(O)O— moieties, wherein one ormore oxygen atoms can be independently replaced by S or NR¹⁷, whereinR¹⁷ is understood to be a C₁₋₉ aliphatic, a

[0085] wherein R¹⁸ is a C₁₋₁₀ unsubstituted or substituted aliphatic, aC₃₋₈ unsubstituted or substituted cycloalkyl, benzyl, phenyl,substituted benzyl or substituted phenyl. When the benzyl or phenyl issubstituted, preferably it is substituted with one or two substituentsselected from the group consisting of halogen, hydroxy, a C₁₋₄ alkyl, aC₁₋₄ alkoxy, an amino, a mono-C₁₋₄ alkylamino, a di-C₁₋₄ alkylamino,phenyl and phenoxy.

[0086] In one embodiment of the invention, X in Formula Ia is aninorganic moiety as described in U.S. Pat. No. 5,212,204. Preferredembodiments of Formula Ia, in which X is inorganic, are ⁻O₃S— (sulfite)and —O⁻ (oxide).

[0087] In another embodiment of the present invention, X in Formula Iais a polyamine as defined in U.S. Pat. No. 5,250,550. Thus, thepolyamine O²-glycosylated diazeniumdiolates have the formula

[0088] wherein Q is the same as the R in Formula Ia and is defined asabove, b and d can be the same or different and are zero or one, R¹, R²,R³, R⁴, and R⁵ are the same or different and are hydrogen, substitutedor unsubstituted C₃₋₈ cycloalkyl, substituted or unsubstituted C₁₋₁₂straight or branched chain alkyl, substituted or unsubstituted benzyl,substituted or unsubstituted benzoyl, substituted or unsubstitutedC₃-C₁₂ olefinic, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,t-butoxycarbonyl, or 2,2,2-tri-halo-t-butoxycarbonyl. The values of i,j, and k in Formula II can be the same or different and are integersfrom 2 to 12.

[0089] In a preferred embodiment of the present invention, thediazeniumdiolates are derived from the compounds disclosed in U.S. Pat.Nos. 5,039,705 (Keefer et al.) and 4,954,526 (Keefer et al.), and, thus,have the formula

[0090] wherein R is the same as the R in Formula Ia and is defined asabove, R¹⁹ and R²⁰ are the same or different and are hydrogen, a C₁-12straight chain alkyl, a C₃₋₁₂ branched chain alkyl, or a C₂₋₁₂ straightor C₃₋₁₂ branched chain olefinic, provided that both R¹⁹ and R²⁰ are nothydrogen. Any of the aforementioned substituents can be unsubstituted orsubstituted with an alkoxy, an acyloxy, an acylthio, a hydroxy, a haloor a benzyl group.

[0091] Alternatively, R¹⁹ and R²⁰, together with the nitrogen atom towhich they are bonded, form a heterocyclic ring selected from the groupconsisting of:

[0092] wherein A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸ ishydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chain alkyl, aC₃₋₈ cycloalkyl, a substituted or an unsubstituted aryl (such as phenyl,tolyl or the like), or carboxylato and derivatives thereof as furtherdescribed herein, and R⁹ is hydrogen, a C₁₋₆ straight chain alkyl or aC₃₋₆ branched chain alkyl. The aforementioned groups can beunsubstituted or substituted as appropriate.

[0093] Exemplary aza crown groups (i.e., where A is N) are1-aza-12-crown-4, 1-aza-15-crown-5, and 1-aza-18-crown-6. Where A isnitrogen, the nitrogen atom, itself, can be substituted, as described,for example, in U.S. patent application Ser. No. 08/475,732.

[0094] Further examples include the O²-glycosylated diazeniumdiolatesderived from the compounds disclosed in U.S. Pat. No. 5,250,550, and,thus, have the formula wherein D is

[0095] and wherein R²¹ is the same as the R in the saccharide of FormulaIa and is defined as above, and R¹⁰ and R¹¹, which can be the same ordifferent, can be any suitable group, examples of which includehydrogen, a C₃₋₈ cycloalkyl, a C₁₋₁₂ straight or branched chain alkyl,benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,t-butoxycarbonyl and 2,2,2-trihalo-t-butoxycarbonyl. In Formula IV, f isan integer from 0 to 12.

[0096] A preferred O²-glycosylated diazeniumdiolate is one in which,with respect to Formula Ia, X is N(CH₂CH₂NH₂)₂ and R is fucose ormannose.

[0097] The above compounds can be prepared in accordance with methodsknown to those of skill in the art. Reagents for glycopyranosylationinclude acetobromo-α- galactose and acetobromoglucosamine. Reagents forglycofuranosylation include tribenzyl-α-arabinofuranosyl bromide andbromoacetylxylose.

[0098] Oligosaccharides are commercially available from, for example,Sigma Chemical Co. (St. Louis, Mo.) and Carbomer Specialty Biochemicalsand Polymers (Westborough, Mass.). In addition, oligosaccharides can besynthesized in accordance with well-established procedures, includingchemical and enzymatic preparation, such as those described inPreparative Carbohydrate Chemistry, Stephen Hanessian, ed., MarcelDekker, New York, N.Y. (1997) and Polysaccharides in MedicinalApplications, Severian Dumitriu, ed., Marcel Dekker,. New York, N.Y.(1996)

[0099] A protected straight- or branched-chain polysaccharide can beactivated toward reaction with the diazeniumdiolate ion by halogenationof the anomeric terminus, followed by glycosylation of thediazeniumdiolate. Activated disaccharides for generation ofO²-glycosylated diazeniumdiolates include acetobromo-α-maltose andacetobromo-α-lactose.

[0100] O²-Glycosylated diazeniumdiolates are useful where molecularsignalling and recognition processes, including cell adhesion, involvecarbohydrates. For example, O²-glycosylated diazeniumdiolates arebelieved to be useful in the treatment of infection, such as that due toa parasite (e.g., leishmania), a virus or a bacterium, as well asinflammation and metastasis. In this regard, an O²-glycosylateddiazeniumdiolate can be prepared so as to be directed to amannose-fucose receptor as exemplified in Example 36. It is believedthat the sugar residue, in this instance mannose, protects thediazeniumdiolate. The mannose binds to the mannose-fucose receptor on amacrophage, and the O²-mannosylated diazeniumdiolate is imported intothe cell, where the sugar residue is cleaved, and NO is released.

1-[(2-CARBOXYLATO) PYRROLIDIN-1-YL]DIAZENIUMDIOLATES

[0101] With respect to the 1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates, the moiety X of Formula Ia can be

[0102] such that the 1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolatescan be structurally represented by the formula:

[0103] wherein R²² is hydrogen, hydroxyl, OM, wherein M is a cation,halo, or X¹R²³R²⁴, wherein X¹ is N, O or S and, when X¹ is N, R²³ andR²⁴ are independently a substituted or an unsubstituted C₁₋₂₄ alkyl,C₃₋₂₄ cycloalkyl, C₂₋₂₄ olefinic, aryl (such as acridine, anthracene,benzene, benzofuran, benzothiophene, benzoxazole, benzopyrazole,benzothiazole, carbazole, chlorophyll, cinnoline, furan, imidazole,indole, isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline,naphthalene, oxazole, phenanthrene, phenanthridine, phenothiazine,phenoxazine, phthalimide, phthalazine, phthalocyanine, porphin,pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrocoline, pyrrole, quinolizinium ion, quinoline, quinoxaline,quinazoline, sydnone, tetrazole, thiazole, thiophene, thyroxine,triazine, and triazole), or heterocyclic group, such as glycosyl, andthe like, and when X¹ is O or S, there is no R²⁴ group. Alternatively,when X¹ is nitrogen, R²³ and R²⁴, together with the nitrogen to whichthey are bonded, form a heterocyclic ring, such as a heterocyclic ringselected from the group consisting of:

[0104] in which A is N, O or S, w is 1 to 12, y is 1 or 2, z is 1 to 5,R⁸, R⁹, R²⁵ and R²⁶ are hydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈branched chain alkyl, a C₃₋₈ cycloalkyl, or an aryl. The aforementionedgroups can be unsubstituted or substituted as appropriate.

[0105] The R²⁶ substituent on the nitrogen (N-4) can be a hydrogen, aC₁₋₈ alkyl group, an aryl group, or C(O)—YR²⁷, wherein Y is sulfur oroxygen, or nitrogen and R²⁷ is CH₂OCH₃, vinyl, a C₁₋₉ straight chainalkyl, a C₃₋₆ branched chain alkyl, a C₃₋₈ cycloalkyl, polyethyleneglycol, polysaccharide, or other polymer, a peptide, or a protein. YR²⁷can be an activating linker, such as a hydroxy succinimidyl group, forlinkage to proteins, peptides, phospholipids, polysaccharides,oligosaccharides, purines, pyrimidines, and biocompatible polymers(i.e., polyethylene glycol, polylactides, and polycaprolactone). YR²⁷can be an activating moiety for the carbonyl group, making the carbonylgroup an electrophilic site that reacts with nucleophilicfunctionalities of oligopeptides, polyamines and proteins. YR²⁷ cancause the carbonyl group to react with many nucleophiles, and can reactwith a polymer, such as polyethylene glycol, to form a polymer-boundcompound.

[0106] Further with respect to the1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates, the moiety R ofFormula Ia can be any covalently bound organic or inorganic moiety,which is other than hydrogen and is a C₁₋₁₂ straight chain or C₃₋₁₂branched chain alkyl, a C₂₋₁₂ straight chain or C₃₋₁₂ branched chainolefinic, a C₁₋₁₂ acyl, sulfonyl, C₃₋₁₂ cycloalkyl, carboxamido, aglycosyl group as described above, an aryl group as described below, ora group of the formula —(CH₂)_(n)—ON═N(O)NR²⁸R²⁹, wherein n is aninteger of 2-8, and R²⁸ and R²⁹ are independently a C₁₋₁₂ straight chainalkyl, a C₃₋₁₂ branched chain alkyl, a C₁₋₁₂ straight chain or C₃₋₁₂branched chain olefinic, or R²⁸ and R²⁹, together with the nitrogen atomto which they are bonded, form a heterocyclic group, preferably apyrrolidino, piperidino, piperazino or morpholino group. Theaforementioned R groups can be unsubstituted or substituted asappropriate. Preferred substitutions include those made with hydroxy,halo, acyloxy, alkoxy, acylthio, or benzyl.

[0107] The above compounds can be prepared in accordance with methodsknown to those of skill in the art. For example, see Sanger, Biochem. J.39: 507-515 (1945).

[0108] O²-Substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates offer advantagesover other diazeniumdiolates in that they are more stable in aqueoussolution than the O²-unsubstituted anion and, in many cases, they can beactivated for NO release by enzymatic action. Furthermore, if anN-nitroso derivative is formed by net formal cleavage of the N—N doublebond of the 1-[(2-carboxylato)pyrrolin-1-yl]diazen-1-ium-1,2-diolate,the N-nitroso compound is noncarcinogenic. Such compounds are believedto be particularly useful in the treatment of fulminant liver failure,malaria, respiratory problems, impotence, and a variety ofcardiovascular/hematologic disorders.

[0109] Polymer Bound Diazeniumdiolates

[0110] Another particularly useful embodiment of the present inventioncomprises O²-aryl diazeniumdiolates of Formula I or O²-glycosylateddiazeniumdiolates of Formula Ia, wherein X is a polymer, or wherein anyO²-aryl diazeniumdiolate or O²-glycosylated diazeniumdiolate of thepresent invention is incorporated into a polymeric matrix. PROLI/NO alsocan be polymer bound—through R²⁰ as well as R. Both of these embodimentsresult in the N₂O₂ ⁻ functional group being “bound to the polymer.” By“bound to a polymer,” it is meant that the N₂O₂ ⁻ functional group isassociated with, part of, incorporated with or contained within thepolymeric matrix physically or chemically.

[0111] Physical association or bonding of the N₂O₂ ⁻ functional group tothe polymer may be achieved by coprecipitation of the polymer with anitric oxide/nucleophile complex as well as by covalent bonding of theN₂O₂ ⁻ group to the polymer. Chemical bonding of the N₂O₂ ⁻ group to thepolymer may be by, for example, covalent bonding of the nucleophilicmoiety of the nitric oxide/nucleophile adduct to the polymer such thatthe nucleophilic residue to which the N₂O₂ ⁻ group is attached formspart of the polymer, itself, i.e., is in the polymer backbone or isattached to pendant groups on the polymer backbone. The manner in whichthe nitric oxide-releasing N₂O₂ ⁻ functional group is associated with,part of, or incorporated with or contained within, i.e., “bound” to thepolymer is inconsequential to the present invention and all means ofassociation, incorporation and bonding are contemplated herein.

[0112] Site-specific application of the polymer-bound adduct compositionenhances the selectivity of action of the nitric-oxide releasing N₂O₂functional group. If N₂O₂ ⁻ functional groups attached to the polymerare necessarily localized, then the effect of their nitric oxide releasewill be concentrated in the tissues with which they are in contact. Ifthe polymer is soluble, selectivity of action can still be arranged, forexample, by linkage to or derivatization of an antibody specific to thetarget tissue. Similarly, linkage of N₂O₂ ⁻ groups to small peptidesthat mimic the recognition sequences of ligands for important receptorsprovides localized nitric oxide release, as would linkage tooligonucleotides capable of site-specific interactions with targetsequences in a nucleic acid.

[0113] The O²-diazeniumdiolates of the present invention can be derivedfrom the materials disclosed in U.S. Pat. Nos. 5,525,357 (Keefer et al.)and 5,405,919 (Keefer et al.), and in U.S. patent application Ser. No.08/419,424 (Smith et al.), each of which is incorporated by reference.Any of a wide variety of polymers can be used in the context of thepresent invention. It is only necessary that the polymer selected isbiologically acceptable. Illustrative of polymers suitable for use inthe present invention are polyolefins, such as polystyrene,polypropylene, polyethylene, polytetrafluorethylene, polyvinyl chloride,polyvinylidene difluoride, and polyethers such as polyethylene glycol,polysaccharides such as dextran, polyesters such aspoly(lactide/glycolide), polyamides such as nylon, polyurethanes,polyethyleneimines, biopolymers such as peptides, proteins,oligonucleotides, antibodies and nucleic acids, starburst dendrimers,polysaccharides, and the like.

[0114] In this regard, a polymer containing a diazeniumdiolate can bereacted with a saccharide, such that the saccharide becomes bound to theN₂O₂ ⁻ functional group.

[0115] Formation of a diazeniumdiolate from a biopolymer provides abiopolymer-bound diazeniumdiolate composition that can be applied withspecificity to a biological site of interest. Site-specific applicationof the biopolymer-bound diazeniumdiolate enhances the selectivity ofaction of the nitric oxide-releasing diazeniumdiolate, which occursfollowing the cleavage of the O²-aryl or O²-glycosylated bond or the O—Rbond in PROLI/NO (see pg. 33). As with the other polymers disclosedabove, if the diazeniumdiolate attached to the biopolymer is localizedbecause of the inherent properties of the molecule, then the effect ofits nitric oxide release will be concentrated in the tissues with whichthey are in contact. If the biopolymer is soluble, selectivity of actioncan still be arranged, for example, by attachment to or derivatizationof an antibody specific to the target tissue. Similarly, linkage ofdiazeniumdiolate groups to small peptides that mimic the recognitionsequences of ligands for important receptors provides localized nitricoxide release, as would linkage to oligonucleotides capable ofsite-specific interactions with target sequences in a nucleic acid.Other proteins, peptides, polypeptides, nucleic acids andpolysaccharides can be similarly utilized. U.S. Pat. No. 5,405,919(Keefer et al.) and U.S. Pat. No. 5,632,981 (Saavedra et al.), herebyincorporated in their entireties by reference, disclose similarcompounds and manufactures useful in the preparation of thediazeniumdiolates.

[0116] By way of illustration, an O²-arylated piperazinediazeniumdiolate can be covalently attached to a polypeptide containingthe IKVAV recognition sequence, which is important in tumor cellchemotaxis. Through retention of both the capacity to regenerate NO asan anti-adhesive agent and the affinity of the IKVAV sequence for tumorcells and/or sites in the vascular and lymphatic systems, where thetumor cells tend to attach, metastasis can be reduced or even prevented.Further, the aryl moiety can be chosen such that it provides additionalantitumor cell activity. Substitutions at the N⁴ position of piperazinecan be used to link the glycosylated diazeniumdiolate to peptides,polypeptides, proteins, polysaccharides and nucleotides.

[0117] It is contemplated that the diazeniumdiolates of the presentinvention can be used to coat prostheses, stents, and medical implants,such as breast implants, prior to surgical connection to the body as ameans of reducing the risk of solid state carcinogenesis associatedtherewith. Additionally, the prostheses and implants can be manufacturedusing a diazeniumdiolate as an integral component of the startingmaterials. Medical devices incorporating a diazeniumdiolate provide aninvaluable two-pronged approach to the treatment of many biologicaldisorders, providing useful medical structures that also advantageouslyprovide local release of NO.

[0118] Compositions

[0119] As is well-known in the art, nitric oxide and compoundscomprising N₂O₂ ⁻ functional groups can have a wide range of utilities,in part because of the multifaceted role of nitric oxide inbioregulatory processes. Accordingly, the present invention alsoprovides a composition, including a pharmaceutical composition,comprising a present inventive diazeniumdiolate. Preferably, thepharmaceutical composition additionally comprises a pharmaceuticallyacceptable carrier.

[0120] One skilled in the art will appreciate that suitable methods ofadministering the diazeniumdiolate compositions of the present inventionto an animal, such as a mammal, are available, and, although more thanone route can be used to administer a particular composition, aparticular route can provide a more immediate and more effectivereaction than another route. Pharmaceutically acceptable carriers arealso well-known to those who are skilled in the art. The choice ofcarrier will be determined, in part, both by the particular compositionand by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations of thepharmaceutical compositions of the present invention.

[0121] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the diazeniumdiolatedissolved in diluents, such as water or saline, (b) capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solids or granules, (c) suspensions in an appropriateliquid, and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible carriers. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to theactive ingredient, such carriers as are known in the art.

[0122] The diazeniumdiolates of the present invention, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

[0123] Formulations suitable for parenteral administration includeaqueous and non-aqueous solutions, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried(lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

[0124] The dose administered to an animal, particularly a human, in thecontext of the present invention should be sufficient to effect atherapeutic response in the animal over a reasonable time frame. Thedose will be determined by the strength of the particular compositionsemployed (taking into consideration, at least, the rate of NO evolution,the extent of NO evolution, and the bioactivity of the decompositionproducts derived from the diazeniumdiolates) and the condition of theanimal, as well as the body weight of the animal to be treated. The sizeof the dose also will be determined by the existence, nature, and extentof any adverse side effects that might accompany the administration of aparticular composition. A suitable dosage for internal administration is0.01 to 100 mg/kg per day. A preferred dosage is 0.01 to 35 mg/kg perday. A more preferred dosage is 0.05 to 5 mg/kg per day. A suitableconcentration of O²-aryl diazeniumdiolates in pharmaceuticalcompositions for topical administration is 0.05 to 15% (by weight). Apreferred concentration is from 0.02 to 5%. A more preferredconcentration is from 0.1 to 3%.

[0125] Methods of Use

[0126] In view of the above, the present invention provides methods orusing a present inventive diazeniumdiolate. In one embodiment, a methodof treating an animal, such as a mammal, with a biological disordertreatable with nitric oxide, is provided. The method comprisesadministering to the animal, e.g., the mammal, an amount of andiazeniumdiolate in accordance with the present invention sufficient totreat the biological disorder in the animal. In this embodiment,“biological disorder” can be any biological disorder, including abiological disorder due to a genetic defect or infection with aninfectious agent, such as a virus, bacterium or parasite, as long as thedisorder is treatable with nitric oxide.

[0127] In another embodiment of a method of use, a method is providedfor treating an animal, such as a mammal, for infection with, forexample, a virus, a bacterium, or a parasite (e.g., leishmania). Themethod comprises administering to the animal, e.g., the mammal, anamount of a diazeniumdiolate sufficient to treat the infection in theanimal.

[0128] In one aspect of this embodiment of the invention, a method isprovided for treating an animal, such as a mammal, for infection with,for example, a virus, such as a retrovirus, in particular HIV, moreparticularly HIV-1, a bacterium, such as a Gram-positive bacterium, or aparasite, such as Giardia, any one of which comprises a zinc fingerprotein that can be inactivated by an O²-aryl diazeniumdiolate. By “zincfinger protein” is meant a protein comprising a short amino acid domaincontaining cysteines alone or cysteine and histidine ligands, both ofwhich coordinate with zinc and interact with nucleic acids (South andSummers, “Zinc Fingers,” Chapter 7, In: Adv. Inorg. Biochem. Ser. 8, pp.199-248 (1990), which is hereby incorporated by reference in itsentirety, including the content of all references cited therein). By“inactivated” is meant partial or complete loss of activity of the zincfinger protein to be inactivated. Such inactivation should not result ininactivation of biologically important zinc finger proteins in theanimal, itself, to such an extent as to compromise unduly the health andwell-being of the animal. The method comprises administering to theanimal, e.g., the mammal, an amount of an O²-aryl diazeniumdiolatesufficient to inactivate the zinc finger protein in said infectiousagent so as to treat the infection in the animal.

[0129] The above-described method also can be adapted as a means oftreating a plant, plant cell or tissue culture thereof for infectionwith an infectious agent, such as a virus, e.g., tobacco streak virus(TSV) or alfalfa mosaic virus (AIMV) (South and Summers (1990), supra;and Sehnke et al., Virology 168: 48 (1989)).

[0130] The methods described herein are useful against zinc fingerscomprising the motif C-X2-C-X4-H-X4-C (see, e.g., Wain-Hobson et al.,Cell 40(1): 9-17 (1985)), in which “C” represents cysteine, “H”represents histidine, “X” represents any amino acid, and the numbers “2”and “4” represent the number of “X” amino acids. Such a motif ischaracteristic of retroviruses, in particular the gag protein ofretroviruses. Accordingly, the methods herein are useful againstretroviruses, such as HIV, and, in particular, HIV-1 (Rice et al.,Nature Medicine 3(3): 341-345 (1997); and Rice et al., Reviews inMedical Virology 6: 187-199 (1986)), which comprises nucleocapsid p7proteins (NCp7 proteins) that include two zinc binding domains. Actualand/or potential zinc fingers also have been identified in, amongothers, the gene products of the EIA genomic region of adenoviruses, thelarge T antigens from simian virus 40 (SV40) and polyoma viruses, theUvrA protein in E. coli (Culp et al., PNAS USA 85: 6450 (1988)), murineleukemia virus (MuLV-F; Green et al., PNAS USA 86: 4047 (1989)), andbacteriophage proteins (Berg, Science 232: 484 (1986)), such as gene 32protein (G32P) from bacteriophage T4 (Giedroc et al., Biochemistry 28:2410 (1989)). Such proteins can be isolated in accordance with methodsknown in the art (see references cited in South and Summers (1990),supra), and the O²-aryl diazeniumdiolates, which can inactivate suchzinc finger proteins, can be identified in accordance, for example, withthe zinc finger assay described herein and in Rice et al., J. Med. Chem.39: 3606-3616 (1996).

[0131] To the extent that steroid hormone receptors comprise zincfingers with motifs comprising 4 or 5 cysteines, an O²-aryldiazeniumdiolate can be used to modulate steroid hormone activity in ananimal, such as a mammal. Accordingly, the present invention alsoprovides a method of modulating steroid hormone activity in an animal,such as a mammal, which is in need of modulation of steroid hormoneactivity and which comprises a steroid hormone receptor proteincomprising a zinc finger that can be inactivated by an O²-aryldiazeniumdiolate. The method comprises administering to the animal,e.g., the mammal, an amount of an O²-aryl diazeniumdiolate sufficient toinactivate the steroid hormone receptor protein so as to modulatesteroid hormone activity in the animal.

[0132] In yet another embodiment, a method for treating an animal, suchas a mammal, for cancer and metastasis thereof is provided. The methodcomprises administering to the animal, e.g., the mammal, an amount ofdiazeniumdiolate sufficient to prevent the growth or metastasis of thecancer in the animal.

[0133] In one aspect of this embodiment, a method for treating ananimal, such as a mammal, for cancer is provided, wherein the cancer isdue, at least in part, directly or indirectly, to the activity of a zincfinger protein that can be inactivated by an O²-aryl diazeniumdiolate.The method comprises administering to the animal, e.g., the mammal, anamount of O²-aryl diazeniumdiolate sufficient to inactivate the zincfinger protein so as to treat the cancer in the animal (Rice et al.,PNAS 89: 7703-7707 (1992)), i.e., prevent the growth or metastasis ofthe cancer in the animal.

[0134] In still yet another embodiment, a method is provided fortreating an animal, such as a mammal, for cancer, wherein the cancer isresistant to treatment with a chemotherapeutic agent (see, e.g., Kelleyet al., Biochem. J. 304: 843-848 (1994)), in particular a DNA damagingagent, such as an alkylating agent or an oxidizing agent, due, forexample, to the action of an enzyme that adversely affects the activityof the chemotherapeutic agent. The method comprises administering to theanimal, e.g., the mammal, an amount of an O²-aryl diazeniumdiolatesufficient to render the cancer in the animal susceptible to treatmentwith the chemotherapeutic agent. Accordingly, such a method can be usedas an adjunct therapy to chemotherapy as needed.

[0135] For example, certain O²-aryl diazeniumdiolates can be synthesizedto fit into the active site of glutathione S-transferase, specificallyisoenzyme π (see, e.g., Ji et al., Biochemistry 32(49): 12949-12954(1993); and Ji et al., Biochemistry 36: 9690-9702 (1997)). Accordingly,inversible consumption or glutathione from the active site ofglutathione S-transferase-π with an O₂-aryl diazeniumdiolate couldprevent the enzyme from detoxifying a variety of xenobiotic compounds,such as chemotherapeutic drugs, especially alkylating agents, such aschlorambucil, melphalan and hepsulfam, and other DNA-damaging agents,such as agents that induce electrophilic attack or oxidization, byenzymatic conjugation of the compound with glutathione (see, e.g.,Morgan et al., Cancer Chemother. Pharmacol. 37: 363-370 (1996)). Thismethod also has applicability to screening drug-resistant cancer celllines in vitro.

[0136] In another embodiment, a method is provided for treating aninanimate object for the presence of a potentially infectious virus,bacterium, or parasite. The method comprises contacting the inanimateobject with an amount of a present inventive diazeniumdiolate sufficientto reduce the presence of the potentially infectious virus, bacterium orparasite. By “potentially infectious” is meant the capability ofinfecting an animal, such as a mammal.

[0137] In one aspect of this embodiment, a method is provided forreducing on an inanimate object the presence of a potentially infectiousagent, such as a virus, a bacterium, or a parasite, any one of whichcomprises a zinc finger protein that can be inactivated by an O²-aryldiazeniumdiolate. The method comprises contacting the inanimate objectwith an amount of an O²-aryl diazeniumdiolate sufficient to inactivatethe zinc finger protein so as to reduce the presence of the potentiallyinfectious agent, e.g., virus, bacterium or parasite, on the inanimateobject. By “potentially infectious” is meant the capability of infectingan animal, such as a mammal, directly or indirectly.

EXAMPLES

[0138] The following examples further illustrate the present inventionand, of course, should not be construed as in any way limiting itsscope. With respect to the following examples, NO was obtained fromMatheson Gas Products (Montgomeryville, Pa.), β- and α-glycosidases andporcine liver esterase were obtained from Sigma Chemical Co. (St. Louis,Mo.), polyurethane (Tecoflex) was obtained from Thermedics Inc. (Woburn,Mass.), and glucose and mannose were obtained from Aldrich Chemical Co.(Milwaukee, Wis.). Proton NMR spectra were recorded with a 300 MHzVarian Unity Plus or a Varian XL-200 NMR spectrometer. Spectra wereobtained in deuterochloroform for covalent compounds and in D₂O forsalts. Chemical shifts are reported in parts per million (ppm) downfieldfrom TMS. Low and high resolution mass spectral (MS) measurements werecarried out on a VG-Micromass Model 7070 spectrometer. Unless otherwiseindicated, MS data were collected in the electron impact mode withsample introduction via direct probe. Ultraviolet (UV) spectra were runas solutions in water or 0.01 M NaOH on an HP 8451A Diode Arrayspectrophotometer. Glutathione S-transferase kinetics were monitored bymeasuring the change in UV absorbance at 380 nm with a Beckman DU 640spectrophotometer. Chemiluminescence measurements were done on a ThermalEnergy Analyzer Model 502A instrument (Thermedics, Inc., Woburn, Mass.).Elemental analyses were performed by Atlantic Microlab Inc.

Example 1

[0139] This Example illustrates the preparation ofO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

[0140] A solution of 1.67 g (11 mmol) of sodiumdiethylaminodiazeniumdiolate in 20 ml of 5% aqueous sodium bicarbonatewas cooled to 0° C. under nitrogen. A solution of 1.3 ml (0.01 mol) of2,4-dinitrofluorobenzene in 10 ml of t-butyl alcohol was added slowly. Aprecipitate formed upon addition. The mixture was allowed to warm up toroom temperature gradually, then stirred overnight. The product wasextracted with dichloromethane and subsequently washed with cold dilutehydrochloric acid followed by sodium bicarbonate solution. The organiclayer was dried over sodium sulfate, filtered through a layer ofmagnesium sulfate, and evaporated under vacuum to give 1.3 g of a redoil, which crystallized on standing. Recrystallization from ethanol gaveyellow-orange needles: m.p. 76-7° C.; NMR δ1.25 (t, 6H), 3.58 (q, 4H),7.68 (d, 1H), 8.44 (m, 2H), 8.89 (m, 1H); UV (ethanol) λ_(max)(ε) 218(17.4 mM⁻¹ cm¹⁻) and 302 (15.6 mM⁻¹ cm⁻¹) nm; MS, exact mass, calculatedfor C₁₀H₁₃N₅O₆: (M+) 299.0865; measured M⁺ 299.08658. Analysis, C, H, N,calculated for: C₁₀H₁₃N₅O₆: C, 40.13%; H, 4.35%; N, 23.41%. Found: C,40.21%; H, 4.43%; N, 23.37%.

Example 2

[0141] This Example illustrates the regeneration of the anionicdiazeniumdiolate from its O²-aryl substituted form(O²-(2,4-dinitrophenyl) 1-(N,N-diethyamino)diazen-1-ium-1,2-diolate).

[0142] A solution of 85 mg (0.28 mmol) of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, prepared as in Example 1,in 1 ml of ether was cooled to −4° C. and treated with 1 ml ofdiethylamine. The solution was kept at −4° C. for 1 hr, giving aprecipitate. The solid was collected by filtration. The filtrate wasconcentrated and analyzed by NMR; the residue proved to be identical toan authentic sample of 2,4-dinitro-N,N-diethylaniline. The precipitatewas washed with petroleum ether and dried under N₂ to give 5.4 mg ofproduct having λ_(max) 250 nm; NMR (D₂O) δ0.96 (t, 6H), 1.28(t, 6H),2.94 (q, 4H), 3.08 (q, 4H). This product proved to be identical to anauthentic sample of diethylammonium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

Example 3

[0143] This Example illustrates the chemical cleavage of the O²-arylbond of an O²-aryl diazeniumdiolate mediated by sodium methoxide.

[0144] A solution of 16 mg (0.064 mmol) of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate in 1 ml of ether wastreated with 29 μl of 25% sodium methoxide in methanol (0.14 mmol) andallowed to stand at −4° C. for 2 hr. The solid precipitate was collectedby filtration, washed with ether and dried under vacuum to yield 4 mg ofa solid identical to an authentic sample of1-(N,N-diethylamino)diazen-1-ium-1,2-diolate sodium salt.

Example 4

[0145] This Example illustrates the kinetics of reaction ofO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate withsodium methoxide in methanol. The kinetics of this reaction show therate of conversion of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate to1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion in alkaline ornucleophilic environments.

[0146] An excess of NaOMe was used in the reactions; aliquots werecollected at intervals and quenched with 0.1 N HCl in methanol. Thedisappearance of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, monitored by HPLC, wasfound to fit the first-order rate equation. This was determined byplotting log[O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate] vs. time to find k_(obs′)at four different concentrations of NaOMe. Similarly, the second-orderrate constant (7.87 M⁻¹ min⁻¹) was determined by plotting log k_(obs)vs. log[NaOMe].

Example 5

[0147] This Example illustrates the preparation ofO²-(2,4-dinitrophenyl) 1-(N-isopropylamino)diazen-1-ium-1,2-diolate.

[0148] A solution of 84 mg (0.597 mmol) of sodium1-(N-isopropylamino)diazen-1-ium-1,2-diolate in 1 ml of 5% sodiumbicarbonate was cooled to 0° C. and 69 mg (0.55 mmol) of2,4-dinitrofluorobenzene was added. The ice bath was removed, themixture was allowed to stir at room temperature overnight, and then themixture was extracted with dichloromethane. The extract was dried oversodium sulfate, filtered and evaporated in vacuo to give 86 mg of afilm, which crystallized on standing: m.p. 92-93° C. NMR δ1.39 (d, 6H),3.99 (septet, 1H), 6.93 (d, 1 H), 8.27 (dd, 1H), 8.5 (b, 1H), 9.15 (d,1H).

Example 6

[0149] This Example illustrates the synthesis of pyrrolidinium1-[pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

[0150] A solution of 36 g (0.507 mol) of pyrrolidine in 50 ml of etherand 25 ml of acetonitrile was placed in a 500 ml Parr bottle, degassedand charged with 40 psi of nitric oxide. The reactor was cooled to −80°C. The pressure was maintained at 40 psi. After 4 hr, the pressure wasreleased, and the crystalline product was collected by filtration in afritted glass funnel and then washed with cold ether under an atmosphereof nitrogen. The material was dried in a vacuum desiccator at 1 mm Hgand 25° C. for 3 hr to give 23 g (45%) of white needles: m.p. 68 70° C.Analysis C,H,N: Calculated for C₈H₁₈N₄O₂: C, 47.51%; H, 8.97%; N,27.70%; Found, C, 47.62%; H, 9.04%; N, 27.46%.

[0151] The pyrrolidinium salt was converted to the more stable sodiumsalt for subsequent O²-arylations by treatment with 10 N NaOH to promotecation exchange. It was then flooded with ether. The product wascollected by filtration.

Example 7

[0152] This Example gives an alternate method of preparing the sodiumsalt of the 1-(pyrrolidin-1-yl)diazen-1-ium1,2-diolate presented inExample 6.

[0153] A solution of 28.2 g (0.397 mol) of pyrrolidine in 100 ml ofacetonitrile and 100 ml of ether was mixed with 94 ml (0.4 mol) of 25%sodium methoxide in methanol. The resulting solution was flushed withnitrogen then charged with 40 psi of NO and stirred at room temperaturefor two days forming a thick precipitate. (The precipitate had begun toform within 1 hr of exposure to NO.) The pressure was released and theproduct was collected by filtration. The product was washed with etherand dried under vacuum to give 32.1 g (54%) of a white powder: UV (0.01N NaOH) λ_(max) (ε), 252 nm (8.84 mM⁻¹ cm⁻¹); t_(1/2) 8.5 set at 25° C.and 2.8 sec at 37° C. in pH 7.4 phosphate buffer; NMR (D₂O) δ1.91 (m,4H), 3.22 (m, 4H).

Example 8

[0154] This Example illustrates the preparation ofO²-(2,4-dinitrophenyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate.

[0155] A solution of 556 mg (3.63 mmol) of sodium1(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate in 10 ml of 5% aqueous sodiumbicarbonate was cooled to 0° C. A solution of 456 μl (3.63 mmol) of2,4-dinitrofluorobenzene in 2 ml of t-butyl alcohol was added and theresulting mixture was stirred at room temperature overnight. Theyellow-orange precipitate was collected by filtration, washed withwater, and dried to give 758 mg of product, which was recrystallizedfrom ethanol: m.p. 94-95° C.; NMR, δ2.04 (m, 4H), 3.35 (m, 4H), 6.90 (d,1H), 8.20 (dd, 1H), 8.67 (d, 1H); MS, m/z(%), 297 (M⁺, 1), 220 (100),237 (30), 190 (94), 180 (15), 162 (10), 149 (26), 130 (20), 100 (95), 70(24), 63 (35), 56 (18). Exact Mass: calculated for C₁₀H₁₁N₅O₆ (M⁺)297.0708; measured 297.0709.

Example 9

[0156] This Example illustrates the preparation of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate.

[0157] A solution of 20 g (0.126 mol) of N-carboethoxypiperazine in 60ml of methanol was placed in a Parr bottle. The solution was treatedwith 27.4 ml (0.126 mol) of 25% sodium methoxide in methanol; the systemwas evacuated, charged with 40 psi of nitric oxide and kept at 25° C.for 48 hr. The white crystalline product was collected by filtration andwashed with cold methanol as well as with copious amounts of ether. Theproduct was dried under vacuum to give a 14.5 g (48%) yield of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate: m.p.184-5° C.; UV (0.01 N NaOH) λ_(max) (ε) 252 nm (10.4 mM⁻¹ cm⁻¹); NMR(D₂O) δ1.25 (t, 3H), 2.15 (q, 2H) 3.11 (m, 4H), 3.68 (m, 4H). Analcalcd. for C₆H₁₃N₄O₄Na: C, 35.00%; H, 5.42%; N, 23.33%; Na, 9.58%.Found: C, 34.87%; H, 5.53%; N, 23.26%; Na, 9.69%.

[0158] The half-life of this compound at pH 7 and 25° C. was assessed at5 min. This measurement was based on the loss of the 252 nm chromophorein the ultraviolet spectrum.

Example 10

[0159] This Example illustrates the preparation ofO²-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate.

[0160] A solution of 1.073 g (0.0045 mol) of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate in 10 ml of5% sodium bicarbonate was cooled at 0° C. under nitrogen. A partialsolution of 0.89 ml (0.0044 mol) of 2,4-dinitrofluorobenzene in 10 ml oft-butyl alcohol was added. A precipitate formed upon addition; themixture was allowed to stir at room temperature for 4 hr. The productwas extracted with dichloromethane. The extracts were washed with water,dried over sodium sulfate and filtered through a layer of anhydrousmagnesium sulfate. Evaporation of the solvent gave an orange glass whichcrystallized on standing. The product was recrystallized fromethanol:dichloromethane to give 1.3 g (76%) of analytically purematerial: m.p. 140-141° C.; NMR δ1.32 (t, 3H), 3.63 (m, 4H), 3.74 (m,4H), 4.19 (q, 2H), 7.66 (d, 1 H), 8.48 (q, 1H), 8.88 (d, 1 H); UV (H₂O)λ_(max) (ε) 210 nm (13.3 mM⁻¹ cm⁻¹), 300 nm (12 mM⁻¹ cm⁻¹). Anal calcd.for C₁₃H₁₆N₆O₈: C, 40.61%; H, 4.20%; N, 21.87%; Found: C, 40.74%; H,4.13%; N, 21.98%.

Example 11

[0161] This Example illustrates the preparation ofO²-(2-chloropyrimidin-4-yl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

[0162] A solution of 600 mg (4 mmol) of 2,4-dichloropyrimidine in 2 mlof dimethylsulfoxide and 5 ml of tetrahydrofuran was added via syringeto a slurry of 678 mg (4.37 mmol) of sodium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate in 5 ml of tetrahydrofuranat room temperature under nitrogen and the resulting mixture was stirredfor 72 hr. Five (5) ml of ether was added to the mixture. After washingwith water, the organic layer was dried over sodium sulfate, filteredthrough a layer of magnesium sulfate, and evaporated to give 679 mg ofan oil which crystallized at −20° C. This material was recrystallizedfrom ether-petroleum ether: m.p. 37-38° C.; NMR δ1.25 (t, 6H); 3.56 (q,4H), 7.00 (d, 1H), 8.50 (d,1H); UV, λ_(max) (ε) 268 nm (9.3 mm⁻¹ cm⁻¹).Analysis C, H, N: Calculated for C₈H₁₂N₅O₂Cl: 39.11%; H, 4.92%; N,28.51%; Cl, 14.43%; Found: C, 38.96%; H, 4.96%; N, 28.35%; Cl, 14.60%.

Example 12

[0163] This Example illustrates the preparationO²-(2-chloropyrimidin-1-yl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate.

[0164] A solution of 262 mg (1.76 mmol) of 2,4-dichloropyrimidine in 3ml of dimethylsulfoxide was added to a slurry of 424 mg (1.76 mmol) ofsodium 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate in10 ml of tetrahydrofuran at room temperature under nitrogen and stirredfor 72 hr. The resulting homogeneous solution was treated with 100 ml ofwater. The precipitate was collected by filtration and dried undervacuum to give 300 mg of product: m.p. 136-137° C.; NMR δ1.29 (t, 3H),3.69 (m, 4H), 3.71 (m, 4H), 4.18 (q, 2 H), 6.99 (d, 1 H), 8.52 (d, 1 H);(UV) λ_(max) (ε) 270 nm (4.1 mM⁻¹ cm⁻¹).

[0165] This compound undergoes nucleophilic substitution with methoxideto displace the chlorine atom at the C2 position and thediazeniumdiolate at the C4 position to give 2,4-dimethoxypyrimidine.

Example 13

[0166] This Example describes the synthesis of the following compounds:

[0167] General synthesis of compounds 1 through 5: A 1 M solution ofsodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate in dimethylsulfoxidewas stirred at 5° C. under nitrogen. A 1 M solution containing 0.95molar equivalents of the arylating agent in tetrahydrofuran was injectedthrough a septum. The reaction mixture was allowed to warm up to roomtemperature, stirred overnight, quenched with ice-water and extractedwith ether. The ether was washed with water, dried over sodium sulfate,filtered through a layer of maganesium sulfate and concentrated on arotary evaporator. The methods of purification varied with eachpreparation and are described with the individual compounds below.(Note: Compounds 1 through 5 are selected products from O²-aryl compoundlibraries built using solution phase synthetic methods in parallelfashion). NMR spectra were run in CDCl₃.

[0168] O²-(2-Nitro-4-trifluoromethylphenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, 1: Arylation was carriedout with 4-fluoro-3-nitrobenzotrifluoride. Purification of the productwas carried out on preparative HPLC using a 1 inch C-18 column elutedwith 20% aqueous acetonitrile with a solvent gradient to 50%acetonitrile: 50% water. A 42% yield of product was obtained as an oil:NMR δ1.23 (t,6H), 3.50 (q,4 H), 7.66 (d, 1 H), 7.82 (d, 1 H), 8.28 (s, 1H).

[0169] O²-(2-Nitro-4-carboxylatophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, 2: 4-Fluoro-3-nitrobenzoicacid was used in this preparation. Purification of the product wascarried out on a Biotage Flash 40 system with a 4.0×15.0 cm KP-Silcolumn. The system was eluted with 5:1 dichloromethane:ethyl acetate at15 psi of air at a rate of elution of 25 ml/min to give a 22% yield ofproduct: mp 115-6° C.; NMR δ1.22 (t, 6 H), 3.33 (q, 4 H), 7.06 (d, 1 H),8.03 (dd, 1 H), 8.37 (m, 1 H).

[0170] O²-(5-Nitropyrid-2-yl)1-(N,N-diethyl)diazen-1-ium-1,2-diolate, 3:The product of reaction with 2-bromo-5-nitropyridine was recrystallizedfrom ether:ethanol to give pure 3 in 62% yield: mp 77-8° C.; NMR δ1.24(t, 6 H), 3.53 (q, 4 H), 7.21 (dd, 1 H), 8.52 (dd, 1 H), 9.17 (dd, 1 H).Analysis C,H,N: Calculated for C₉H₁₃N₅O₄: C, 42.35%; H, 5.13%; N,27.44%; Found: C, 42.46%; H, 5.14%; N, 27.52%.

[0171]O²-(3,5-Dinitropyrid-2-yl)1-(N,N-diethyl)diazen-1-ium-1,2-diolate, 4:Anylation was effected with 2-chloro-3,5-dinitropyridine as described inthe general procedure. The crude product was recrystallized fromether:petroleum ether to give 4 in 33% yield: mp 56-7° C.; NMR δ1.28 (t6 H), 3.57 (q,4 H), 8.81 (d, 1 H), 9.10 (d, 1 H).

[0172] O²-(3-Nitropyrid-2-yl)1-(N,N-diethyl)diazen-1-ium-1,2-diolate, 5:2-Chloro-3-nitropyridine was used in this reaction. The crude productwas purified on a Flash 40 system using a 4.0×7.0 cm KP-Sil columneluted with 100% dichloromethane to give a 52% yield of product as aviscous oil: NMR δ1.25 (t, 6 H), 3.55 (q, 4 H), 7.26 (m, 1 H), 8.48 (m,2 H).

Example 14

[0173] This Example illustrates the preparation of O²-vinyl1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO).

[0174] To 3.56 g (9.2 mmol) of O²-(2-bromoethyl)1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate 2-bromoethylester was added 10 ml of 10 N sodium hydroxide solution.

[0175] The two-phase mixture was stirred at 25° C., whereupon thecompound gradually dissolved in the aqueous layer. After stirringovernight, the UV of the reaction mixture exhibited an absorptionmaximum at 266 nm (starting material absorbed at 252 nm), indicating theformation of a vinyl group.

[0176] The solution was cooled to 0° C. and carefully acidified to pH 4by the slow addition of 10% hydrochloric acid. Care must be taken tokeep the solution cold while acid is added. The acidic solution wasextracted with ethyl acetate, dried over sodium sulfate and filteredthrough a layer of magnesium sulfate. Evaporation of the solvent gave1.4 g of an oil. Purification was carried out on a Flash 40 System(Biotage) using a 4.0×7.0 cm KP-Sil column and 2:1 ethylacetate:cyclohexane as the eluant:ir (film) 3163, 2987, 1734, 1630, 1490cm⁻¹; NMR (CDCl₃) δ2.06-2.3 (m,4H), 3.62 (m,2H), 4.47 (q,1H), 4.77 (ABq,1H), 5.02 (ABq, 1H), 6.75 (q,1H)) UV λ_(max) (ε) 266 nm(6.3 mM⁻¹ cm⁻¹);MS,m/z(%)201 (M⁺,5), 176(10), 150(49), 145(27), 114(9), 99(45),70(99.9), 69(57), 68(45).

Example 15

[0177] This Example illustrates the regeneration of NO fromO²(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate inthe presence, but not the absence, of glutathione.

[0178] A solution containing 1 mM glutathione (GSH) in 10 mM phosphatebuffer was degassed by purging with argon for 10 min, whereupon a 3 mlaliquot was mixed with 3 μl of a dioxane solution that was 2 mM inO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate. NOrelease was monitored by chemiluminescence while the mixture was held at37° C. After a brief lag time, peak nitric oxide generation was observedat approximately 15 minutes after the reaction was initiated andcontinued at readily detectable levels for approximately 100 minutes.Total NO generation during the first 112 min was approximately 9 nmol.Assuming that 2 nmol of NO is generated per mol ofO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate,this 9 nmol corresponds to roughly 75% of the theoretical yield.

[0179] When the reaction was repeated as above but with exclusion of theGSH, no NO generation was observed. The nucleophilic glutathione reactedwith the O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate to produce NO according tothe equation shown below.

[0180] This example is illustrative of the ability of some of theO²-aryl diazeniumdiolate compounds of the present invention to undergonucleophilic substitution by nucleophilic side-chains of amino acidssuch as cysteine, which are often found in the active sites of enzymes.The result of such nucleophilic substitution is the generation of anaryl derivative of the displacing amino acid residue and adiazeniumdiolate capable of producing NO, through a predictable,first-order reaction.

[0181] O²-(2,4-Dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate and glutathione were alsoassayed in the presence and absence of glutathione S-transferase. Assayswere conducted in a thermostated cell compartment at 25° C., using 0.1 Mphosphate buffer at pH 7.4, with a final volume of 3 ml. Theconcentration of the enzyme was 0.7 μg/ml, whereas that of glutathionewas 1.4 mM. The concentration of diazeniumdiolate was varied from 50-100μM. Using the integrated form of the Henri-Michaelis-Menten equation,K_(m) was found to be 46.3 μM and V_(max) was found to be 0.89 μM min⁻¹.

Example 16

[0182] This Example illustrates a route of synthesis which is useful inthe production of diazeniumdiolated nucleotides, nucleosides, andnucleic acids and further illustrates a route to synthesis of O²-aryldiazeniumdiolates, which comprises converting an amino group to adiazonium group, followed by reaction of the diazonium group with adiazeniumdiolate.

[0183] 2′-Deoxycytidine is reacted with nitric oxide in the presence ofa suitable 1-electron oxidant which results in the conversion of theamino group of the cytidine into a diazonium group while reducing theoxidant and producing hydroxide ion. The resulting diazotized (i.e.,diazonium derivatized) pyrimidine is then reacted with1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion, as described in theprevious examples, to generate a diazeniumdiolated 2′-deoxyuridinederivative. This diazeniumdiolated 2′-deoxyuridine derivative can bereacted with strong nucleophiles (e.g., hydroxide ions). This willresult in the regeneration of1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion plus 2′-deoxyuridine.This regenerated 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion willgenerate NO in a predictable, first-order reaction. This Exampledemonstrates a basis for a mechanism that is suitable for targetingnitric oxide to a particular site of a mammalian body, so that thespecificity of NO action can be increased.

Example 17

[0184] This example demonstrates the ability of an O²-aryldiazeniumdiolate to inactivate a zinc finger protein by zinc ejection.

[0185] Samples of recombinant nucleocapsid protein p7 (p7NC) from HIV-1(L. O. Arthur, AIDS Vaccine Program, NCI-FCRDC, Frederick, Md.) wereprepared at μg/ml in 10 mM sodium phosphate buffer (pH=7.0) and treatedwith 25 μmol of an O²-aryl diazeniumdiolate in a total volume of 1.0 ml.At various time intervals, as shown in FIG. 1, which is a graph of Trp37fluorescence (RFU) versus time (min), the samples were diluted {fraction(1/10)} in 10 mM sodium phosphate buffer (pH=7.0) to preventintroduction of any artifactual quenching effects and the fluorescenceintensity of the tryptophan residue (Trp37) in the C-terminal zincfinger of p7NC in each sample was determined as previously described(Rice et al., Int. Antiviral News 3: 87-89 (1995)). The excitation andemission wavelengths utilized with a Shimadzu RF5000 spectrofluorimeterwere 280 and 351 nm, respectively. The results are shown in FIG. 1, inwhich ◯ represents the negative control, i.e., no drug, □ represents thepositive control, i.e., 642151 (see Rice et al. (1997), supra), ▪represents the compound of Example 1 (LK1), ♦ represents the compound ofExample 8 (LK2), ▴ represents the compound of Example 5 (LK3), •represents the compound of Example 10 (LK4), and x represents thecompound of Example 11 (LK5). The results indicate that an O²-aryldiazeniumdiolate can eject zinc from a zinc finger protein.

Example 18

[0186] This example demonstrates the anti-HIV activity of O²-aryldiazeniumdiolates.

[0187] The tumor cell line of T4 lymphocytes designated CEM-SS was grownin a synthetic medium with fetal bovine serum (Rice et al., Advances inPharmacol. 33: 389-438 (1995)). O²-aryl diazeniumdiolates wereadministered to HIV-1-infected and uninfected CEM-SS cells atconcentrations ranging from 10^(−3.5) to 10^(−7.0) M in accordance withthe XTT-based cell viability assay of the National Cancer Institute(see, e.g., Rice et al. (1995), supra).

[0188] After exposure of CEM-SS cells to the compounds, the percentageof T-cell viability was assessed. The viability of HIV-1-infected CEM-SScells, which were contacted with a subtoxic concentration of any one ofthe above-described O²-aryl diazeniumdiolates, was substantially,increased in comparison to untreated cells. Compounds 1-3 from Example13 were especially effective.

Example 19

[0189] This example describes the preparation of disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

[0190] A solution of 10 g (0.087 mol) of L-proline in 39 ml (0.18 mol)of 25% sodium methoxide in methanol, 20 ml of methanol and 40 ml ofether was degassed and exposed to 40 psi of nitric oxide for 20 hr. Thepressure was released and the solid residue was collected by filtration,washed with ether and dried under vacuum to give 17 g of a white solid:mp 250° C. (dec.); UV (0.01 N NaOH) λ_(max) (ε) 252 nm (8.4 mM⁻¹ cm⁻¹);NMR (D₂O) δ1.71 (m, 1H), 1.91 (m, 2H), 2.27 (m, 1H), 3.27-3.43 (m, 2H),4.04 (m, 1H) (a methanol singlet at 3.34 is also observed); ¹³C NMR,24.45 ppm, 30.97, 48.73 (methanol), 54.95, 67.70, 182.75.

[0191] Anal. C,H,N: Calculated for, C₅H₇N₃O₄Na₂.CH₃OH, C, 28.69%; H,4.41%; N, 16.73%; Na, 18.30%; Found C, 28.65%; H, 3.99%; N, 16.74%; Na,18.04%.

Example 20

[0192] This example describes the preparation of O²-methyl1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate methyl ester.

[0193] Disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (methanolsolvate, FW 251; 6.8 g; 0.027 mol) was placed in a 300 ml 3-neck flaskand cooled to −20° C. Cold methanol (−20° C.; 200 ml) was added to thesolid while stirring to give a homogeneous solution, which was cooledfurther to −35° C. A solution of 9.5 ml (0.1 mol) of dimethylsulfate in25 ml of ether was added dropwise over a 15 min period. The reactionmixture was then allowed to warm to room temperature gradually andstirred for an additional 4 hr. The progress of the reaction wasmonitored on silica gel TLC using 10:1 dichloromethane:ethyl acetate asthe eluant. The reaction mixture was filtered, the methanol was removedon a rotary evaporator, and the residue was extracted withdichloromethane. The solution was washed with aqueous sodiumbicarbonate, dried over sodium sulfate and filtered through a layer ofmagnesium sulfate. Evaporation of the solvent gave an oil, whichcrystallized on standing. Recrystallization from ether:petroleum ethergave 945 mg (18%) of an analytically pure sample: mp 62-63° C.; UV (0.01N NaOH), λ_(max) (ε) 252 nm (6.79 mM⁻¹ cm⁻¹); NMR δ2.05 (m, 3H), 2.30(m, 1H), 3.65 (m, 1H), 3.75 (s, 3H), 3.83 (m, 1H), 3.96 (s, 3H), 4.55(m, 1H); MS m/z (%) 203 (M⁺, 6), 188 (20), 58 (35), 120 (22), 99 (100),95 (34), 69 (36), 59 (24); exact mass calculated for C₇H₁₃N₃O₄ (M⁺)203.0906, found (M⁺) 203.0906.

[0194] Anal. C,H,N: Calculated for C₇H₁₃N₃D₄, C, 41.38%; H, 6.45%; N,20.68%: Found C, 41.48%; H, 6.43%; N, 20.59%.

Example 21

[0195] This example describes the preparation ofO²-(N,N-dimethylsulfamoyl)1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

[0196] A solution of 1.08 ml (0.01 mol) of N,N-dimethylsulfamoylchloride in 5 ml of tetrahydrofuran was added dropwise to a cold (0° C.)solution of 1.57 g (0.0062 mol) of disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate in 25 ml of0.1 N NaOH in saline solution. The reaction mixture was allowed to warmup to room temperature and stirred overnight. The aqueous layer wasextracted with dichloromethane and the organic layer was dried overanhydrous sodium sulfate. The aqueous layer showed no significant UVabsorption after extraction and, thus, indicated that the extractionproducts were devoid of diazeniumdiolate. The organic layer was filteredthrough a layer of magnesium sulfate and the solvent was removed on arotary evaporator to give 989 mg of a pale yellow oil, which waschromatographed on silica gel using 5:1 dichloromethane:ethyl acetate asthe eluant. The fractions containing the desired product were combinedand concentrated under vacuum to give a solid, which was recrystallizedfrom ether-petroleum ether: mp 97-98° C.; UV (0.01 N NaOH) λ_(max) (ε)266 nm (8.05 mM⁻¹ cm⁻¹); NMR δ2.16, (m, 3H), 2.40 (m, 1H), 3.01 (s, 6H),3.83 (m, 1H), 3.94 (m, 1H), 4.69 (q, 1H), 6.80 (b, 1H).

[0197] Anal. C,H,N,S: Calculated for C₇H₁₄N₄SO₆, C, 29.79%; H, 5.00%; N,19.85%; S, 11.36%: Found C, 29.93%; H, 5.09%; N, 19.76%; S, 11.27%.

Example 22

[0198] This example describes the preparation of O²-methoxymethyl1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate methoxymethylester.

[0199] A slurry of 485 mg (1.93 mmol) of disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate in 20 ml ofanhydrous tetrahydrofuran was cooled to 0° C. under a nitrogenatmosphere. Triethylamine (0.5 ml) was added to the cold solutionfollowed by the slow addition of 360 mg (4.45 mmol) ofchloromethylmethyl ether and a subsequent dropwise addition of 0.5 ml ofmethanol. The solution was then stirred in the cold for 1.5 hr. Thereaction mixture was allowed to warm up to room temperature and stirredunder nitrogen for an additional 1.5 hr. The reaction was quenched withcrushed ice, whereupon the solvent was removed on a rotary evaporatorand the residue was extracted with dichloromethane. The organic phasewas washed with water, dried over sodium sulfate, filtered throughmagnesium sulfate and evaporated in vacuo to give 330 mg of a yellowoil, which was purified on a silica gel column with 5:1dichloromethane:ethyl acetate as the eluant: UV (H₂O) λ_(max) (ε) 250 nm(8.58 mM⁻¹ cm⁻¹); NMR δ2.09 (m, 3H); 2.35 (m, 1H), 3.48 (s, H), 3.71 (m,2H), 3.90 (m, 1H), 4.61 (dd, 1H), 5.17 (ab q, 2H), 5.31 (ab q, 2H).

[0200] Anal. C,H,N: Calculated for C₉H₁₇N₃O₆: C, 41.06%; H, 6.51%; N,15.96%: Found C, 40.87%; H, 6.53%; N, 15.76%.

Example 23

[0201] This example describes the preparation of O²-(2-bromoethyl)1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate 2-bromoethylester.

[0202] A solution of 20 ml (0.28 mol) of bromoethanol in 50 ml ofdichloromethane was cooled to 0° C. and 11.25 ml (0.28 mol) of sulfurylchloride in 50 ml of dichloromethane was added dropwise to the solution.The resulting solution was kept at 4° C. for 72 hr. The solution waswashed with cold 10% NaOH until the washings tested distinctly basic.The organic layer was dried over sodium sulfate, filtered through alayer of magnesium sulfate and concentrated on a rotary evaporator. Theresulting crude product (2-bromoethoxysulfonyl chloride, BrCH₂CH₂OSO₂Cl)was vacuum-distilled to give 35 g (56%) of a colorless oil: bp 73-75° C.at 1.5 mmHg; NMR δ3.64 (t, 2H), 4.752 (t, 2H); MS m/z (%) 221 (M⁺, 1),143 (10), 129 (25), 106 (100), 93 (62). Analysis C,H,N,S,X: Calculatedfor C₂H₄SO₃ClBr: C, 10.75%; H, 1.80%; S, 14.35%; total halogen as Br,71.52%; and as Cl, 31.72%; Found: C, 10.82%; H, 1.80%; S, 14.35%; totalhalogen as Br, 71.63%; and as Cl, 31.78%.

[0203] Disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (4.86 g;0.0194 mol) was placed in a 100 ml round-bottom flask, together with 2.2g of anhydrous sodium carbonate. The flask was immersed in a dryice-acetonitrile bath (at −40° C.) and 50 ml of cold (−20° C.) ethanolwas added. Then the mixture was stirred and allowed to stabilize at −40°C. under an atmosphere of nitrogen. To the cold slurry was added, via asyringe, 9.45 g (0.0422 mol) of 2-bromoethoxysulfonyl chloride over aperiod of 10 min. After stirring for 2 hr, the reaction mixture wasallowed to warm to 15° C. and stirred for an additional 2 hr. Thereaction mixture was poured into 250 ml of ice-water and extracted withdichloromethane. The organic layer was washed with aqueous sodiumbisulfite solution, dried over sodium sulfate and filtered through alayer of magnesium sulfate, whereupon the solvent was removed on arotary evaporator. The crude product was chromatographed on a silica gelcolumn using 1:1 cyclohexane:ethyl acetate as the eluant to give 2.7 g(36%) of a pale yellow oil: NMR δ2.11 (m, 3H), 2.35 (m, 1H), 3.55 (m,4H), 3.68 (m, 1H), 3.86 (m, 1H), 4.46 (m, 4H), 4.59 (m, 1H); UV (H²O)λ_(max) (ε) 252 nm (6.6 mM⁻¹ cm⁻¹).

Example 24

[0204] This example describes the preparation ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester.

[0205] 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (1.03 g; 0.0068 mol) wasadded to a solution of 1.33 g (0.0034 mol) of O²-(2-bromoethyl)1-[(2-carboxylato)pyrrolidin-1- yl]diazen-1-ium-1,2-diolate 2-bromoethylester in 35 ml of tetrahydrofuran and the resulting solution was stirredat room temperature under nitrogen. Two equivalents of thiolacetic acid(0.479 ml, 0.0068 mol) were added and the mixture was stirred at roomtemperature for 2 hr. The mixture was filtered and the solid residue waswashed with ether. The filtrate was evaporated to dryness under reducedpressure and the residue was extracted with methylene chloride. Theorganic solution was subsequently washed with ice-cold 5 N HCl, sodiumbicarbonate solution and water. The solution was dried over sodiumsulfate, filtered through a layer of magnesium sulfate and evaporated invacuo to give 710 mg of a yellow oil. Chromatography was carried out ona silica gel column eluted with 1:1 cyclohexane:ethyl acetate: UV (H₂O)λ_(max) (ε) 232 nm (7.0 mM⁻¹ cm⁻¹); NMR δ2.09 (m, 3H), 2.36 (m, 1H),2.38 (s, 6H), 3.09 (m, 4H), 3.78 (m, 2H), 4.27 (m, 4H), 4.55 (m, 1H).

Example 25

[0206] This example describes the determination of the halflife of theImpound produced in Example 24 in the absence and presence of porcineliver esterase at 25° C. and pH 7.4.

[0207] A 0.009 M ethanolic stock solution ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercapto-ethyl)] ester was prepared. The decay of thiscompound was monitored at 25° C. as 1.5×10⁻⁴ M solutions in a 4 mlquartz cuvette containing 3 ml of phosphate buffer (pH 7.4) and 50 ml ofstock solution. The decay of the 232 nm chromophore was monitored on theultraviolet spectrophotometer. The halflife was estimated as 3.2 hr.

[0208] A second set of experiments was carried out using the aboveparameters to measure the decay after addition of 5 ml of porcine liveresterase suspension. The half-life for the esterase reaction was 8 minat 25° C.

Example 26

[0209] This example describes the preparation of a nitricoxide-releasing polymer blend of O²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester.

[0210] A solution of 50 mg (0.132 mmol) ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester in 1 ml of tetrahydrofuran wasdissolved in a solution of 498 mg of polyurethane in 10 ml oftetrahydrofuran. The homogeneous lacquer was concentrated under a streamof dry nitrogen followed by further drying under high vacuum to give asolid, which contained 0.091 mg (0.24 mmol) ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester per mg of polymer composite. Rates ofNO release were measured as a function of time after immersing a 32 mgaliquot of the diazeniumdiolate in 2 ml of phosphate buffer, pH 7.4, at37° C., with a chemiluminescence detector. A set of experiments wascarried out in plain buffer, while another set was done in the presenceof porcine liver esterase. A very small amount of NO was released in theabsence of enzyme over a 200 hr period, while a significant rate of NOproduction was observed when the enzyme was present in the buffer. Thisindicates that as the diazeniumdiolate oozes out of the polymercomposite, it is hydrolyzed by the enzyme with further cleavage to NO.

Example 27

[0211] This example describes the introduction of the nitricoxide-releasing O²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester into β-cyclodextrin.

[0212] β-Cyclodextrin (228 mg, 0.201 mmol) was mixed with 2 ml of waterand heated to 65° C. to give a homogeneous solution. To the warmsolution was added 76 mg (0.201 mmol) of O²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester. Upon mixing, a white precipitateformed. The mixture was allowed to cool to room temperature and theproduct was collected by filtration, washed with water, and dried undervacuum to give 170 mg of product. An aqueous solution containing 33 mgof the O²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)] ester: β-cyclodextrin mixture exhibited anabsorbance maximum at 232 nm and a molar absorptivity (ε) of 10.8mM⁻¹cm⁻¹. Rates of NO release were measured as a function of time afterimmersing a 13 mg aliquot of the encapsulated material in 4 ml ofphosphate buffer, pH 7.4, at 37° C., with a chemiluminescence detector.A set of experiments was carried out in plain buffer, while another setwas done in the presence of porcine liver esterase. A very small amountof NO was released in the absence of enzyme over a 400 hr period while asignificant rate of NO production was observed when the enzyme waspresent in the buffer.

Example 28

[0213] This example describes a general procedure for the preparation ofO²-glycosylated diazeniumdiolates.

[0214] 2,3,4,6-Tetraacetyl-α-D-glucopyranosyl bromide(acetobromoglucose) was prepared as described in Redemann et al., Org.Syn. Coll. Vol. III: 11-14 (1955).2,3,4,6-Tetraacetyl-α-D-mannopyranosyl bromide (acetobromomannose) wasprepared as described in Levene et al., J. Biol. Chem. 90: 247-250(1931). Then, a slurry of 1 eq of a diazeniumdiolate indimethylsulfoxide (DMSO) (0.5 mmol solid/1 ml of DMSO) was stirred with0.03 eq of silver oxide at room temperature under nitrogen. A 0.5 Msolution of 1.2 eq of acetobromomannose or acetobromoglucose in DMSO wasinjected dropwise and the mixture was stirred for three days. Theresulting homogeneous solution was poured into 100 ml of ice-water andextracted with ether. The ether layer was washed with water, dried oversodium sulfate and treated with charcoal. The solution was filteredthrough magnesium sulfate, concentrated on a rotary evaporator, anddried under vacuum. The glucose derivatives were purified byrecrystallization, while the glassy mannose adducts required columnchromatography.

Example 29

[0215] This example describes the preparation of sodium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (“DEA/NO”).

[0216] A solution of 119 g (1.63 mol) of diethylamine in 100 ml of 1:1ether:acetonitrile was placed in a 500 ml Parr bottle. The solution wasdegassed, charged with 40 psi of nitric oxide, and allowed to stand atroom temperature overnight. The pressure was released and thecrystalline product was collected by filtration and dried under nitrogento give 13 g of diethylammonium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate. The salt was treated with10 ml of 10 M sodium hydroxide solution and the resulting paste wastreated with 200 ml of ether to give the sodium salt. The sodium salt(“DEA/NO”) was collected by vacuum filtration, washed with ether, anddried under vacuum to give 7.1 g of product: UV (in 0.01 N NaOH) λ_(max)(ε) 250 (6.88 mM⁻¹cm⁻¹); NMR (D₂O) δ0.96 (t, 3 H), 2.94 (q, 2 H); inDMSO-d₆ δ0.84 (t, 3 H) and 2.75 (q, 2 H).

Example 30

[0217] This example describes the preparation ofO²-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

[0218] DEA/NO (2.98 g; 0.019 mol) in DMSO was reacted withacetobromoglucose (6.9 g; 0.017 mol) as described in the generalprocedure of Example 28. The product was recrystallized from petroleumether to give 5.7 g (72%) 108 mg of a crystalline solid: mp 107-108° C.;UV λ_(max) (ε) 228 nm (6.92 mM⁻¹cm⁻¹); NMR δ1.11 (t, 6 H, J=7.11), 2.02(s, 3 H), 2.03 (s, 3 H), 2.04 (s, 3 H), 2.07 (s, 3 H), 3.21 (q, 4 H,J=7.12), 3.81 (m, 1 H), 4.20 (m, 2 H), 5.14 (m, 1 H), 5.33 (m, 3 H).Anal. Calcd for C₁₈H₂₉N₃O₁₁: C, 46.65; H, 6.31; N, 9.07. Found: C,46.73; H, 6.26; N, 9.01.

Example 31

[0219] This example describes the deacylation ofO²-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate(from Example 30).

[0220] A solution of 253 mg (0.55 mmol) of the above compound in 5 ml ofmethanol was stirred with 10 μl of 25% methanolic sodium methoxide. Theprogress of the reaction was monitored by TLC using 5:1 CH₂Cl₂:ethylacetate. The reaction was complete within 1 h at 25° C.

[0221] Dowex-50W-H⁺ resin (1 g) was added to the stirring methanolicsolution. The mixture was filtered to remove the resin, and themethanolic solution was evaporated under vacuum to give 122 mg (75%) ofO²-glucopyranosyl 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate: UVλ_(max) (ε) 228 nm (6.4 mM⁻¹ cm⁻¹); NMR (CDCl₃) δ1.08 (t,6H), 3.23(9,4H), 5.59 (m,4H), 3.88 (m,2H), 5.29 (m,1H).

[0222] Surprisingly, the deacetylated product cleaved to the1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) anion, then to NO,only extremely slowly at pH 3, despite its acetal-like structure. Evenmore surprisingly, the cleavage proceeded extremely rapidly at pH 13.

Example 32

[0223] This example describes the preparation of sodium1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate.

[0224] A solution of 20 g (0.126 mol) of carboethoxy piperazine in 60 mlof methanol was placed in a Parr bottle. The solution was treated with27.4 ml (0.126 mol) of 25% sodium methoxide in methanol. The system wasevacuated, charged with 40 psi of nitric oxide and kept at 25° C. for 48hr. The white crystalline product was collected by filtration and washedwith cold methanol as well as with copious amounts of ether. The productwas dried under vacuum to give 14.5 g (48% yield) of sodium1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate: mp:184-185° C.; UV (0.01 N NaOH) λ_(max) (ε) 252 nm (10 mM⁻¹ cm⁻¹); NMR(D²O) δ1.25 (t, 3 H), 3.11 (m, 2 H), 3.68(m, 2 H); 2.15 (q, 2 H). Analcalcd. for C₆H₁₃N₄O₄Na: C, 35.00%; H, 5.42%; N, 23.33%; Na, 9.58%.Found: C, 34.87%; H, 5.53%; N, 23.26%; Na, 9.69%. The half-life of thiscompound at pH 7 and 25° C. was estimated as 5 min. This measurement wasbased on the loss of the 252 nm chromophore in the ultraviolet spectrum.

Example 33

[0225] This example describes the preparation of O²-(glucopyranos-2-yl)1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolatetetraacetate ester.

[0226] Acetobromoglucose (2.055 g; 0.005 mol) and 1.11 g (0.00466 mol)of sodium 1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolatewere reacted as described above to give 624 mg (25%) ofO²-(glucopyranos-2-yl)1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolatetetraacetate ester: UV λ_(max) (ε) 228 nm (7.20 mM⁻¹ cm⁻¹); NMR δ1.26(t, 3 H), 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3 H), 2.09 (s, 3 H), 3.46(m, 4H), 3.68 (m, 4 H), 3.82 (m, 1 H), 4.17 (q, 2 H), 4.25 (m, 3 H),5.27 (m, 3 H).

Example 34

[0227] This example describes the preparation of O²-(mannopyranos-2-yl)1-[(1-ethoxycarbonyl)piperazin-4-yl)]diazen-1-ium-1,2-diolatetetraacetate.

[0228] Acetobromomannose (10.2 g ; 0.025 mol) and 5.28 g (0.022 mol) ofsodium 1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate werereacted as described above to give 6.4 g (53%) of a glass: UV λ_(max)(ε) 238 nm (7.5 mM⁻¹ cm⁻¹); NMR δ1.29 (t, 3 H), 2.01 (s, 3H), 2.05 (s,3H), 2.11 (s, 3 H), 2.17 (s, 3 H), 3.13 (m, 1 H), 3.50 (m, 4 H), 3.78(m, 5 H), 4.19 (q, 2 H), 4.27(m, 3 H), 5.28 (m, 3 H), 5.42 (m, 1H).

Example 35

[0229] This example describes the preparation of an O²-glycosylateddiazeniumdiolate directed to a mannose-fucose receptor.

[0230] Bis-[2-(N-ethoxycarbonylamino)ethyl]amine: A three-neck flaskequipped with two dropping funnels was immersed in an ice-water bath.Diethylenetriamine (10.7 g, 0.104 mol) was placed in the cold flask anddissolved in 100 ml of 95% ethanol. To the cold solution was added 10 ml(0.205 mol) of ethylchloroformate, dropwise. A solution of 10.6 g (0.1mol) of sodium carbonate in 100 ml of distilled water was addedsimultaneously with 10 ml (0.205 mol) of ethylchloroformate. Thereaction mixture was allowed to stir at room temperature overnight. Theethanol was removed on a rotary evaporator and the aqueous portion wasextracted with dichloromethane. The organic layer was washed with water,then extracted with 5% hydrochloric acid. The organic layer containingthe neutral products was separated and set aside. The aqueous layer waswashed with dichloromethane and made basic with sodium hydroxide. Theproduct was extracted into dichloromethane, dried over sodium sulfate,filtered through magnesium sulfate and evaporated to give 4 g of acolorless oil: NMR (CDCl₃) δ1.25 (t, 6H), 2.78 (m, 4H), 3.36 (m, 4H),4.14 (q, 4H), 5,13 (b, 2H).

[0231] Sodium1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolate: Asolution of 2.6 g (0.011 mol) ofbis-[2-(N-ethoxycarbonylamino)ethyl]amine in 20 ml of ether and 5 ml ofmethanol was placed in a 50 ml Parr bottle, treated with 2.4 ml (0.011mol) of 25% methanolic sodium methoxide, degassed, cooled to −80° C. andcharged with 50 psi of nitric oxide. A thick precipitate was observedafter 3 hr of stirring. The mixture was exposed to NO for 24 hr, thepressure was released, and the product was collected by filtration. Thesolid was washed with ether and dried under vacuum to give 1.26 g (35%)of the diazeniumdiolate: mp 170-2° C.; UV λ_(max) (ε) 252 nm (7.6 mM⁻¹cm⁻¹); NMR δ1.24 (t, 6H), 3.19 (m, 8H), 4.11 (q, 4H).

[0232] O²-(Mannos-2-yl)1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolatetetraacetate: A partial solution of 251 mg (0.763 mmol) of sodium1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolate in2 ml of dimethylsulfoxide (DMSO) was cooled to 0° C. under nitrogen. Tothis was added 10 mg (0.06 mmol) of silver acetate, followed by the slowaddition of 1 ml of a 0.82 M solution of acetobromomannose intetrahydrofuran. The reaction mixture was allowed to stir at roomtemperature for 48 hr. poured over ice-water, and extracted with ether.The ether solution was dried over sodium sulfate, filtered through alayer of magnesium sulfate, and evaporated under vacuum to give 307 mgof an oil: UV λ_(max) 240 nm.

[0233]O²-(Mannos-2-yl)1-[bis(2-aminoethyl)amino]diazen-1-ium-1,2-diolate]:

[0234] A solution of 145 mg (0.23 mmol) of O²-(mannos-2-yl)1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolatetetraacetate in a mixture of 0.2 ml of 10 N NaOH, 2 ml of ethanol and 2ml of water was heated at reflux for 15 hr. The solution wasconcentrated under vacuum and the remaining aqueous solution wasextracted with dichloromethane. The aqueous solution was evaporated todryness under vacuum. The residue was taken up in methanol, put througha 10 g, 60 cc prepacked C-18 column, and eluted with methanol. Thefractions exhibiting an absorption maximum at 236 nm were combined andevaporated to give 32 mg of a white powder: NMR (CD₃OD) δ2.74 (t, 4H),3.02 (t, 4H) 3.74 (m, 4H), 4.2 (m, 3H); UV λ_(max) 238 nm.

Example 36

[0235] This example describes the preparation of a combinatorial libraryusing disodium 1-(2-carboxylato)pyrrolidin-1-yl diazen-1-ium-1,2-diolate(PROLI/NO) as starting material.

[0236] The piperazine trityl resin 1, available fromCalbiochem-Novabiochem Int'l. (San Diego, Calif.), is treated withsulfuryl chloride to form the chlorosulfonamide 2. Reaction of thisresin with PROLI/NO gives compound 3. The free carboxylic acid can beactivated to 4 by reaction with dicyclohexyl carbodiimide (DCC) andN-hydroxysuccinimide. Nucleophilic addition of R³⁰XH (X═O, N, S) to theresin-bound diazeniumdiolate provides a potentially large library ofcompounds, 5, substituted at the carboxylato portion of the molecule.Base hydrolysis of 5 frees the anionic diazeniumdiolate 6 from theresin. This library, 6, may now be reacted with electrophiles R³¹X toform new sets of compound having structure 7.

[0237] All publications, patents and patent applications, cited hereinare hereby incorporated by reference to the same extent as if eachpublication were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

[0238] While this invention has been described with emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that the preferred embodiments may be varied. It is intendedthat the invention may be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. A polymer comprising a polymeric backbone and atleast one functional group having the formula:

wherein X is selected from the group consisting of an amino, apolyamino, a C₁-C₂₄ aliphatic, a C₆-C₃₀ aryl, a C₃-C₃₀ heteroaromatichaving 1 to 3 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur, a C₃-C₃₀ nonaromatic cyclic, and an oximyl, and Qis selected from the group consisting of an acridinyl, an anthracenyl, abenzimidazolyl, a benzisoxazolyl, a benzofuryl, a benzothienyl, abenzoxazolyl, a benzopyrazolyl, a benzothiazolyl, a carbazolyl, achlorophyllyl, a cinnolyl, a furyl, an imidazolyl, an indolyl, anisobenzofuryl, an isoindolyl, an isoxazolyl, an isothiazolyl, anisoquinolyl, a naphthyl, an oxazolyl, a phenanthryl, a phenanthridinyl,a phenothiazinyl, a phenoxazinyl, a phenyl, a phthalimidyl, aphthalazinyl, a phthalocyaninyl, a pteridinyl, a purinyl, which isoptionally part of a nucleic acid, a ribosylpurinyl, a pyrazinyl, apyrazolyl, a pyridazyl, a pyridyl, a pyrimidyl, which is optionally partof a nucleic acid, a ribosylpyrimidyl, a pyrrocolinyl, a pyrryl, aquinolyl, a quinoxalinyl, a quinazolinyl, a sydnonyl, a tetrazolyl, athiazolyl, a thienyl, a thyroxinyl, a triazinyl, and a triazolyl,wherein a ring atom of Q is bonded to an oxygen atom of the at least onefunctional group, wherein X is bound to the polymeric backbone, andwherein X and Q are optionally substituted.
 2. The polymer of claim 1,wherein the polymeric backbone is selected from the group consisting ofa polyolefin, a polyurethane, a peptide, a protein, a polyethyleneimine,a starburst dendrimer, a polyamide, a polyether, a polyester, a nucleicacid, and a polysaccharide.
 3. The polymer of claim 2, wherein thepolyolefin is selected from the group consisting of a polystyrene, apolyethylene, a polytetrafluoroethylene, a polyvinylchloride, and apolyvinylidene difluoride.
 4. The polymer of claim 2, wherein thepolyamide is nylon.
 5. The polymer of claim 1, wherein Q is part of avitamin.
 6. The polymer of claim 1, wherein Q is part of a hormone. 7.The polymer of claim 1, wherein Q is a pyrimidyl, which, optionally, ispart of a nucleic acid.
 8. The polymer of claim 7, wherein Q is aribosylpyrimidyl.
 9. The polymer of claim 1, wherein Q is a purinyl,which, optionally, is part of a nucleic acid.
 10. The polymer of claim9, wherein Q is a ribosylpurinyl.
 11. The polymer of claim 1, wherein Xis linked to the N¹ nitrogen through an atom other than a carbon atom.12. The polymer of claim 1, wherein X is substituted with one or moremoieties selected from the group consisting of —[N(NO)O⁻], a halo,hydroxy, an alkylthio, an alkoxy, an aryloxy, an amino, cyano, asulfonato, mercapto, nitro, a C₁-C₁₂ aliphatic, a C₃-C₈ cycloalkyl, aC₃-C₈ heterocyclic, a C₂-C₁₂ olefinic, benzyl, phenyl, benzylcarbonyl,phenylcarbonyl, glucosyl, ribosyl, glucosyl, mannosyl, deoxyribosyl,dextranyl, starch, glycogenyl, lactosyl, fucosyl, galactosyl, fructosyl,glucosaminyl, galactosaminyl, heparinyl, maltosyl, sucrosyl, sialyl,cellulose, phosphorylated pentosyl, polyphosphorylated pentosyl,phosphorylated hexosyl, polyphosphorylated hexosyl, phosphono,phosphato, and phosphato in which one or more oxygen atoms areindependently replaced with S or NR¹, wherein R¹ is a C₁-C₈ aliphatic, aC₃-C₈ cycloalkyl, a C₆-C₈ aryl, or a C₃-C₈ heteroaromatic having 1 to 3heteroatoms selected from the group consisting of oxygen, nitrogen andsulfur.
 13. The polymer of claim 1, wherein Q is substituted with one ormore moieties selected from the group consisting of X[N(O)NO]⁻, whereinX is as defined in claim 1, halo, hydroxy, alkylthio, arylthio, alkoxy,aryloxy, amino, mono- or di-substituted amino, ammonio, substitutedammonio, nitroso, cyano, sulfonato, mercapto, nitro, oxo, a C₁-C₂₄aliphatic, a C₂-C₁₂ olefinic, a C₃-C₂₄ cycloalkyl, a C₃-C₂₄heterocyclic, benzyl, phenyl, substituted benzyl, substituted phenyl,benzylcarbonyl, phenylcarbonyl, glucosyl, ribosyl, glucosyl, mannosyl,deoxyribosyl, dextranyl, starch, glycogenyl, lactosyl, fucosyl,galactosyl, fructosyl, glucosaminyl, galactosaminyl, heparinyl,maltosyl, sucrosyl, sialyl, cellulose, phosphorylated pentosyl,polyphosphorylated pentosyl, phosphorylated hexosyl, polyphosphorylatedhexosyl, substituted benzylcarbonyl, substituted phenylcarbonyl,phosphono, phosphato, and phosphato in which one or more oxygen atomsare independently replaced with S or NR¹, wherein R¹ is a C₁-C₁₀aliphatic, a C₃-C₁₀ cycloalkyl, a C₆-C₁₀ aryl, or a C₁-C₁₀heteroaromatic having 1 to 3 heteroatoms selected from the groupconsisting of oxygen, nitrogen and sulfur.
 14. The polymer of claim 1,wherein the functional group has the formula:

wherein b and d can be the same or different and may be zero or one, R¹,R², R³, R⁴, and R⁵ are the same or different and are selected from thegroup consisting of hydrogen, C₃-C₈ cycloalkyl, C₁₋₁₂ straight orbranched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl, and2,2,2-trihalo-t-butoxycarbonyl, and i, j, and k are the same ordifferent and are integers from 2 to
 12. 15. The polymer of claim 1,wherein the functional group has the formula: wherein D is

and wherein R¹⁰ and R¹¹ are the same or different and are selected fromthe group consisting of hydrogen, C₃₋₈ cycloalkyl, C₁₋₁₂ straight orbranched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl, and2,2,2-trichloro-t-butoxycarbonyl, and f is an integer from 0 to
 12. 16.The polymer of claim 1, wherein the functional group has the formula:

wherein R⁶ and R⁷ can be the same or different and are H, a C₁-C₁₂straight chain alkyl, a C₁-C₁₂ alkoxy or acyloxy substituted straightchain alkyl, a C₂-C₁₂ hydroxy or halo substituted straight chain alkyl,a C₃-C₁₂ branched chain alkyl, a C₃-C₁₂ hydroxy, halo, alkoxy, oracyloxy substituted branched chain alkyl, a C₂-C₁₂ straight chainolefinic, or a C₃-C₁₂ branched chain olefinic, wherein R⁶ and R⁷ areoptionally substituted with hydroxy, alkoxy, acyloxy, halo or benzyl; orR⁶ and R⁷ together with the nitrogen atom to which they are bonded forma heterocyclic ring selected from the group consisting of:

wherein A is N, O, or S, w is 1 to 12, y is 1 or 2, z is 1 to 5, R⁸ ishydrogen, a C₁-C₈ straight chain alkyl, a C₃-C₈ branched chain alkyl, aC₃-C₈ cycloalkyl, a C₆-C₃₀ aryl, or a C₃-C₃₀ heteroaromatic having 1 to3 heteroatoms selected from the group consisting of oxygen, nitrogen andsulfur, and R⁹ is hydrogen, a C₁-C₆ straight chain alkyl or a C₃-C₆branched chain alkyl.
 17. The polymer of claim 16, wherein R⁶ ishydrogen.
 18. The polymer of claim 16, wherein R⁶ and R⁷ are ethyl and Qis selected from the group consisting of:


19. The polymer of claim 1, wherein the ring atom of Q bonded to theoxygen atom is carbon or nitrogen.
 20. A composition comprising apolymer of claim 1 and a carrier.
 21. A compound having the formula:

wherein X is selected from the group consisting of an amino, apolyamino, a C₁-C₂₄ aliphatic, a C₆-C₃₀ aryl and a C₆-C₃₀ non-aromaticcyclic, and R is a saccharide, which is attached to the O² of thecompound by the 2 position of a pyranose ring or a furanose ring, withthe proviso that, when X is a saccharide, it is not attached to N¹ ofthe compound by the 2 position of a pyranose or a furanose ring.
 22. Thecompound of claim 21, wherein the saccharide is selected from the groupconsisting of a monosaccharide, a disaccharide, an oligosaccharide, anda polysaccharide.
 23. The compound of claim 22, wherein the disaccharideis sucrose or maltose.
 24. The compound of claim 22, wherein themonosaccharide is mannose, fucose or glucose.
 25. The compound of claim21, wherein said saccharide is a recognition sequence forreceptor-mediated cellular phenomena.
 26. The compound of claim 21,wherein X is an amino group that is linked to the N¹-nitrogen through anitrogen atom.
 27. The compound of claim 21, wherein said X issubstituted with a moiety selected from the group consisting of—[N(NO)O⁻], a halo, a hydroxy, an alkylthio, an alkoxy, an aryloxy, anamino, a cyano, a sulfonato, a mercapto, a nitro, a C₁-C₁₂ aliphatic, aC₃-C₈ cycloalkyl, a C₃-C₈ heterocycloalkyl, a C₃-C₁₂ olefinic, a benzyl,a phenyl, a benzylcarbonyl, a phenylcarbonyl, a saccharide, phosphono,phosphato, and phosphato it which one or more oxygen atoms isindependently replaced with S or NR¹, wherein R¹ is a C₁-C₈ aliphatic, aC₃-C₈ cycloalkyl, benzyl, phenyl, or R¹⁸C═N(OH) in which R¹⁸ is a C₁-C₁₀aliphatic.
 28. The compound of claim 21, having the formula

wherein b and d can be the same or different and may be zero or one, R¹,R², R³, R⁴, and R⁵ are the same or different and are selected from thegroup consisting of hydrogen, C₃-C₈ cycloalkyl, C₁₋₁₂ straight orbranched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-tri-halo-t-butoxycarbonyl, and i, j, and k are the same ordifferent and are integers from 2 to
 12. 29. The compound of claim 21,wherein X is R¹⁹R²⁰N— and R¹⁹ and R²⁰ are the same or different and arehydrogen, a C₁₋₁₂ straight chain alkyl, a C₃₋₁₂ branched chain alkyl, ora C₂₋₁₂ straight or C₃₋₁₂ branched chain olefinic, wherein R¹⁹ and R²⁰are optionally substituted with an alkoxy, an acyloxy, an acylthio, ahydroxy, a halo or a benzyl group, or R¹⁹ and R²⁰, together with thenitrogen atom to which they are bonded, form a heterocyclic ringselected from the group consisting of:

wherein A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸ ishydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chain alkyl, aC₃₋₈ cycloalkyl, an aryl, or carboxylato, and R⁹ is hydrogen, a C₁₋₆straight chain alkyl or a C₃₋₆ branched chain alkyl.
 30. The compound ofclaim 29, wherein R¹⁹R²⁰N— is N(CH₂CH₂NH₂)₂ and the saccharide is fucoseor mannose.
 31. The compound of claim 21, wherein the saccharide isribose, deoxyribose, lactose, galactose, fructose, glucosamine, glucose,mannose, fucose, galactosamine, and glucuronic acid, or a pentose orhexose, either one of which is phosphorylated, 3,5-cyclophosphorylated,or polyphosphorylated.
 32. The compound of claim 31, wherein theglucosamine has the structure:

wherein R¹² and R¹³ can be the same or different and are a hydrogen, aC₁₋₆ alkyl, an acyl, a phosphate, a sulfate, a peptide or a protein. 33.The compound of claim 31, wherein the glucuronic acid has the structure:

wherein R¹⁴ is X¹R¹⁵R¹⁶, wherein X¹ is N, O or S and, when X¹ is N, R¹⁵and R¹⁶ are independently hydrogen or a C₁₋₂₄ alkyl, C₃₋₂₄ cycloalkyl,C₂₋₂₄ olefinic, a C₃-C₃₀ aryl or a heterocyclic group selected from thegroup consisting of:

wherein A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸ ishydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chain alkyl, aC₃₋₈ cycloalkyl, an aryl or carboxylato, and R⁹ is hydrogen, a C₁₋₆straight chain alkyl or a C₃₋₆ branched chain alkyl, and when X¹ is O orS, there is no R¹⁶ group.
 34. The compound of claim 33, wherein the arylis selected from the group consisting of an acridine, an anthracene, abenzene, a benzofuran, a benzothiophene, a benzoxazole, a benzopyrazole,a benzothiazole, a carbazole, a chlorophyll, a cinnoline, a furan, animidazole, an indole, an isobenzofuran, an isoindole, an isoxazole, anisothiazole, an isoquinoline, a naphthalene, an oxazole, a phenanthrene,a phenanthridine, a phenothiazine, a phenoxazine, a phthalimide, aphthalazine, a phthalocyanine, a porphin, a pteridine, a purine, apyrazine, a pyrazole, a pyridazine, a pyridine, a pyrimidine, apyrrocoline, a pyrrole, a quinolizinium ion, a quinoline, a quinoxaline,a quinazoline, a sydnone, a tetrazole, a thiazole, a thiophene, athyroxine, a triazine, and a triazole.
 35. A compound having theformula:

wherein X is an inorganic moiety and R is a saccharide, which isattached to the O² atom of the compound by the 2 position of a pyranosering or a furanose ring.
 36. The compound of claim 35, in which X is⁻O₃S— or ⁻O—.
 37. A compound having the formula: wherein D is

and wherein R²¹ is a saccharide, which is attached to the O² atom of thecompound by the 2 position of a pyranose ring or a furanose ring, f isan integer from 0 to 12, and R¹⁰ and R¹¹, which can be the same ordifferent, can be hydrogen, a C₃-C₈ cycloalkyl, a C₁-C₁₂ straight- or aC₃-C₁₂ branched-chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl or2,2,2-trihalo-t-butoxycarbonyl.
 38. A compound having the formula:

wherein R is a C₁₋₁₂ straight chain alkyl, a C₃₋₁₂ branched chain alkyl,a C₂₋₁₂ straight chain or a C₃₋₁₂ branched chain olefinic, a C₁₋₁₂ acyl,sulfonyl, carboxamido, a glycosyl group, a C₁-C₃₀ aryl group or a groupof the formula —(CH₂)_(n)—ON═N(O)NR²⁸R²⁹, wherein n is an integer of2-8, and R²⁸ and R²⁹ are independently a C₁₋₁₂ straight chain alkyl, aC₃₋₁₂ branched chain alkyl, or a C₂₋₁₂ straight chain or a C₃₋₁₂branched chain olefinic, or R²⁸ and R²⁹, together with the nitrogen atomto which they are bonded, form a heterocyclic group selected from thegroup consisting of a pyrrolidino, a piperidino, a piperazino and amorpholino group; and R²² is hydrogen, hydroxyl, OM, wherein M is acation, a halo, X¹R²³R²⁴, wherein X¹ is O, N or S, and R²³ and R²⁴ areindependently a C₁₋₂₄ alkyl, a C₃₋₂₄ cycloalkyl, a C₂₋₂₄ olefinic, aC₃-C₃₀ aryl, or a heterocyclic group, and, when X¹ is O or S, there isno R²⁴.
 39. The compound of claim 38, wherein R is substituted with ahydroxy, halo, acyloxy, alkoxy, acylthio or benzyl.
 40. The compound ofclaim 38, wherein, when X¹ is nitrogen, R²³ and R²⁴, together with thenitrogen to which they are bonded, form a heterocyclic ring selectedfrom the group consisting of:

wherein A is O, N or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸, R⁹, R²⁵,and R²⁶ are hydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chainalkyl, a C₃₋₈ cycloalkyl, or a C₃-C₃₀ aryl.
 41. The compound of claim38, wherein the aryl is selected from the group consisting of anacridine, an anthracene, a benzene, a benzofuran, a benzothiophene, abenzoxazole, a benzopyrazole, a benzothiazole, a carbazole, achlorophyll, a cinnoline, a furan, an imidazole, an indole, anisobenzofuran, an isoindole, an isoxazole, an isothiazole, anisoquinoline, a naphthalene, an oxazole, a phenanthrene, aphenanthridine, a phenothiazine, a phenoxazine, a phthalimide, aphthalazine, a phthalocyanine, a porphin, a pteridine, a purine, apyrazine, a pyrazole, a pyridazine, a pyridine, a pyrimidine, apyrrocoline, a pyrrole, a quinolizinium ion, a quinoline, a quinoxaline,a quinazoline, a sydnone, a tetrazole, a thiazole, a thiophene, athyroxine, a triazine, and a triazole.
 42. The compound of claim 40,wherein, when X¹ is nitrogen and R²³ and R²⁴, together with the nitrogento which they are bonded, form the heterocyclic ring

R²⁵ is hydrogen, a C₁-C₈ straight chain alkyl, a C₃-C₈ branched chainalkyl, a C₃-C₈ cycloalkyl or a C₃-C₃₀, aryl, R²⁶ is hydrogen, a C₁-C₈alkyl, a C₃-C₃₀ aryl, or C(O)—YR²⁷, wherein Y is sulfur, oxygen ornitrogen and R²⁷ is CH₂OCH₃, vinyl, a C₁-C₉ straight chain alkyl, aC₃-C₆ branched chain alkyl, a C₃-C₈ cycloalkyl, polyethylene glycol, apolysaccharide, a peptide or a protein.
 43. A composition comprising acompound of claim 21 and a carrier.
 44. A pharmaceutical compositioncomprising a compound having the formula:

wherein X and R are saccharides which are attached to the O² atom of thecompound by the 2 position of a pyranose ring or a furanose ring.
 45. Acomposition comprising a compound of claim 35 and a carrier.
 46. Acomposition comprising a compound of claim 37 and a carrier.
 47. Acomposition comprising a compound of claim 38 and a carrier.
 48. Thecompound of claim 21, wherein X and Q are imidazoles.
 49. The compoundof claim 21, wherein X is bound to a polymer.
 50. A method of treatingor preventing a biological disorder in an animal, wherein said disorderis selected from the group consisting of angina, acute myocardialinfarction, congestive heart failure, hypertension and metastasis, whichmethod comprises administering to said animal an amount of a polymer ofclaim 1 sufficient to treat or prevent the biological disorder in saidanimal.
 51. The method of claim 48, wherein said biological disorder isdue to hypertension.
 52. The method of claim 48, wherein said biologicaldisorder is due to acute myocardial infarction.
 53. The method of claim48, wherein said biological disorder is due to metastasis.
 54. A methodof treating or preventing a biological disorder in a mammal, whereinsaid disorder is selected from the group consisting of angina, acutemyocardial infarction, congestive heart failure, hypertension andmetastasis, which method comprises administering to the animal a polymerof claim 1 in an amount sufficient to treat or prevent the biologicaldisorder.
 55. A method of treating an animal infected with an infectiousagent comprising a zinc finger protein that can be inactivated by nitricoxide, the method comprising administering to said animal an amount of apolymer of claim 1 sufficient to inactivate the zinc finger protein inthe infectious agent so as to treat the infection in the animal.
 56. Amethod of treating an animal for cancer, wherein the cancer involves azinc finger protein that can be inactivated by nitric oxide, the methodcomprising administering to the animal an amount of a polymer of claim 1sufficient to inactivate the zinc finger protein so as to treat thecancer in said animal.
 57. A method of treating an animal for cancer,wherein the cancer is resistant to treatment with a chemotherapeuticagent, the method comprising administering to the animal an amount of apolymer of claim 1 sufficient to render the cancer in the animaltreatable with the chemotherapeutic agent.
 58. A method of modulatingsteroid hormone activity in a mammal, wherein the mammal is in need ofmodulation of steroid hormone and wherein the animal has a steroidhormone receptor comprising a zinc finger protein which can beinactivated by nitric oxide, the method comprising administering to theanimal an amount of a polymer of claim 1 sufficient to inactivate thesteroid hormone receptor protein so as to modulate steroid hormoneactivity in the mammal.
 59. The method of claim 55, wherein theinfectious agent is a virus.
 60. The method of claim 59, wherein thevirus is HIV.
 61. The method of claim 55, wherein said infectious agentis a parasite.
 62. The method of claim 61, wherein said parasite isGiardia.
 63. The method of claim 57, wherein the chemotherapeutic agentis a DNA-damaging agent.
 64. The method of claim 63, wherein theDNA-damaging agent is selected from the group consisting of analkylating agent and an oxidizing agent.
 65. A method of treating aplant, a plant cell or a tissue culture thereof, which is infected withan infectious agent comprising a protein that can be inactivated by apolymer of claim 1 which method comprises contacting said plant, plantcell or tissue culture thereof with an amount of a polymer of claim 1sufficient to inactivate the protein in said infectious agent so as totreat the infection in said plant, plant cell or tissue culture thereof.66. The method of claim 65, wherein said infectious agent is a virus.67. The method of claim 66, wherein said virus is selected from thegroup consisting of tobacco streak virus and alfalfa mosaic virus.
 68. Amethod of reducing on an inanimate object the presence of an infectiousagent comprising a zinc finger protein that can be inactivated by apolymer of claim 1, which method comprises contacting said inanimateobject with an amount of a polymer of claim 1 sufficient to inactivatethe zinc finger protein so as to reduce the presence of the infectiousagent on said inanimate object.
 69. The method of claim 68, wherein saidinfectious agent is selected from the group consisting of a virus, abacterium, and a parasite.